Process for preparing mammalian T cell growth factor P40

ABSTRACT

The present invention relates generally to a T cell growth factor. More particularly, the present invention relates to a T cell growth factor which comprises a glycoprotein which supports interleukin 2- and interleukin 4-independent growth of helper T cells especially from murine and human sources and further which is capable of augmenting proliferation of IL3- or IL4-responsive cells. Even more particularly, the present invention relates to the helper T cell growth factor P40, pharmaceutical compositions thereof, antibodies thereto and recombinant DNA clones thereof. The present invention also contemplates a method for inducing the proliferation of helper T cells as well as IL3- and Il4-responsive cells. The helper T cells growth factor contemplated herein is useful in the stimulation of specific cells in the immune system, either alone or in combination with IL3 or IL4.

This application is a Divisional of Ser. No. 07/462,158 filed Jan. 8,1990, now U.S. Pat. No. 5,587,302 which in turn is acontinuation-in-part of Ser. No. 07/408,155, filed Sep. 15, 1989, nowU.S. Pat. No. 5,157,112, which in turn is a continuation-in-part of Ser.No. 07/246,482, filed Sep. 19, 1988, now U.S. Pat. No. 5,208,218.

FIELD OF THE INVENTION

The present invention relates generally to a T cell growth factor. Moreparticularly, the present invention relates to a mammalian T cell growthfactor which is a glycoprotein capable of supporting interleukin 2- andinterleukin 4-independent growth of helper T cells. This factor isfurther capable of augmenting proliferation of IL3- and IL4-responsivecells. Even more particularly, the present invention relates to thehelper T cell growth factor P40, pharmaceutical compositions thereof,antibodies thereto, and nucleic acid encoding P40. The present inventionalso contemplates a method for inducing the proliferation of helper Tcells as well as IL3- and IL4-responsive cells. The helper T cell growthfactor contemplated herein is useful in the stimulation of specificcells in the immune system.

BACKGROUND OF THE INVENTION

Many cytokines are polypeptides which directly or indirectly mediatehost defense mechanisms and/or which mediate tissue growthdifferentiation. Cytokines have been recognized which mediate hostdefense against cancer and/or infection. Such cytokines include theinterferons (IFN-α, IFN-β and IFN-γ), tumor necrosis factor (TNF-α),lymphotoxin (TNF-β), the interleukins (IL1, 2, 3, 4, 5 and 6),leukoregulin, natural killer cell cytotoxic factor (NKCF), transforminggrowth factor (TGF), colony stimulating factors (CSF) such as macrophage(M-CSF), granulocyte (G-CSF) and macrophage, granulocyte-CSF (G,M-CSF)and oncostatin M. Each of the aforementioned cytokines have uniquecharacteristics and a unique range of antiproliferative, cytostatic,antiviral or growth regulatory activity.

Several cytokines are synthesized by leukocytes commonly in response tostimulation by microorganisms, antigens or mitogens. This has beenobserved in vitro. Following this stimulation in cell culture, thesupernatant fluid is retrieved and cytokine activity identified,isolated and further characterized. In recent years, it has becomeincreasingly clear the IL2 is not the only factor controlling T cellgrowth. Indeed, several cytokines, including IL4 (Fernandez-Botran etal., Proc. Natl. Acad. Sci. USA 83: 9689-9693, 1986; Lichtman et al.,Proc. Natl. Acad. Sci. USA 84: 824-827, 1987) G,M-CSF (Woods et al., J.Immunol. 138: 4293-4297, 1987); Kupper et al., J. Immunol. 138:4288-4292, 1987) and, in a human system, the combination of IL1 and IL6(Houssiau et al., Eur. J. Immunol. 18: 653-656, 1988), have now beenshown to induce IL2-independent T cell proliferations. Consequently, theregulation of T cell growth is more complex then originally thought,although IL2 is a potent and broadly active T cell growth factor.

An important subset of T cells is the helper T cell (T_(H)). At leasttwo types of helper T cells have been identified on the basis offunctional criteria. One type of T_(H) cell (T_(H) 1) helps B cells in alinked, antigen-specific manner, and is required early in the response.Another type of T_(H) (T_(H) 2) helps B cells in a nonlinked manner andis required later in the response.

Several years ago, a collection of helper T cell lines from lymph nodesof antigen-primed mice was obtained using the procedure described byCorradin et al., J. Immunol. 119: 1048-1053, 1977. These cell lines wereinitiated by culture in the presence of antigen and were subsequentlymaintained, without addition of exogenous growth factors, by regularfeeding with antigen and irradiated splenic antigen-presenting cells.Most of these cells produce large amounts of IL3, IL4, IL5 and IL6, butno IL2 and, therefore, belong to the T_(H) 2 type defined by Mosmann etal., J. Immunol. 136: 2348-2357, 1986.

In accordance with the present invention, it is surprisingly discoveredthat two clones derived from the above-mentioned cell lines proliferatedin response to their own conditioned medium in the absence of antigenand feeder cells. The subject invention relates to a novel T cell growthfactor distinct from other known cytokines. The new growth factor isuseful as a therapeutic compound to stimulate proliferation of helper Tcells.

SUMMARY OF THE INVENTION

The present invention is directed to a mammalian T cell growth factorwhich supports interluekin 2-independent and interleukin 4-independentgrowth of helper T cells, and is preferably obtained from mouse or humansources.

More particularly, this T cell growth factor is a protein having theidentifiable characteristics of P40, derivatives or fragments thereofand the further capability of augmenting proliferation of IL3- andIL4-responsive cells. Methods of isolating P40, its derivatives andfragments are also provided.

Another aspect of the present invention relates to a pharmaceuticalcomposition comprising an effective amount of P40, a derivative orfragment thereof and a pharmaceutically acceptable carrier useful in thestimulation of specific cells in the immune system. Optionally, thesecompositions may also contain IL3 or IL4.

Still another aspect of the present invention relates to antibodiesspecific to P40, or an antigenic derivative or an antigenic fragmentthereof, useful in diagnostic assays for P40.

Yet another aspect of the present invention relates to a recombinant DNAmolecule and expression vectors encoding the polypeptide portion ofmammalian P40, a derivative or a fragment thereof, thereby providing aconvenient source of recombinant P40.

Still yet another aspect of the present invention contemplates a methodof proliferating helper T cells which comprises incubating said cellswith a proliferating effective amount of P40 or a derivative thereof fora time and under conditions sufficient for said cells to proliferate.

A still further aspect of this invention relates to a method ofproliferating IL3- or IL4-responsive cells by administering acombination of P40 and IL3 or IL4 to a mammal, especially a human, for atime and under conditions sufficient to stimulate said cells toproliferate.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphical representation depicting long-termantigen-independent T cell growth induced by helper T cell supernatant(SN). TUC2.15 cells are grown without feeder cells and antigen in normalmedium (□), in medium supplemented with IL2 (20 U/ml, ▪) or withoTUC2.15 SN (5% v/v, ▴).

FIG. 2 is a graphical representation depicting purification of P40.TUC7.51 supernatants is fractioned sequentially on an Ultrogel AcA65 gelfiltration column (A), a TSK-phenyl hydrophobic interaction column (B),a Mono-Q anion exchange column (C) and C1-reversed phase column (D). Theshaded area represents P40 activity. Molecular mass standards shown inpanel A are bovine serum albumin (BSA, 67 kDa), natural IL5 (45 kDa) andrecombinant mouse IL6 (22 kDa).

FIG. 3 is a graphical representation depicting growth factor activity ofpurified P40. TS1 cells (3×10³ cells/well) are cultivated in thepresence of increasing doses of purified P40. After 3 days, cellsnumbers are evaluated by measuring hexosaminidase levels.

FIG. 4 illustrates the purity of P40 and its extent of glycosylation.Panel A is a photograph showing silver-stained NaDodSo₄ /PAGE ofpurified P40. The sample is run under reducing conditions. Panel B is anautoradiograph of ¹²⁵ I-P40 treated with various glycosylases. Mr ofstandards is given kDa.

FIG. 5 is a graphic illustration of the sequencing strategy of themurine P40 gene.

FIG. 6 is a graphic illustration of the expression strategy of themurine P40 gene.

FIG. 7 shows the amino acid sequence of murine P40 obtained by chemicalsequencing and the various peptides used in obtaining this sequence.

FIG. 8 is a graphic illustration of the separation of endoproteinaseAsp-N peptides of Cm-P40 by RP-HPLC.

FIG. 9 is a graphic illustration of a multi-wavelength plot of the HPLCprofile of FIG. 8 using a photodiode array detector at wavelengths of(A) 290 nm, (B) 280 nm, (C) 254 nm and (D) 215 nm.

FIG. 10 is a graphic illustration of a derivative spectral analysis ofendoproteinase Asp-N peptide D1 with the zero other spectrum indicatedby (---) and the second order spectrum indicated by ( ).

FIG. 11 is a graphic illutration of the microbore RP-HPLC separation ofpeptides of Cm-P40 derived from digestion with various proteases. PanelA shows the peptides from S. aureus V8 protease digestion of Cm-P40endoproteinase Asp-N peptide D3. Panel B shows the peptides from achymotrypsin digestion of Cm-P40. Panel C shows the peptides from atrypsin digestion of Cm-P40.

FIG. 12 is a graphic illustration of the elution profile of the blockedamino terminal peptide D1 and related synthetic peptides on RP-HPLC. Zindicates pyroglutamic acid.

FIG. 13 depicts a restriction cleavage map and the exon/intronorganization of the human and mouse P40 genes. Closed boxes representcoding regions, and numbers indicate their respective length in basepairs. Open box corresponds to 5' untranslated region. Arrows representdirection and extent of sequence analysis. Restriction endonucleasesites used for sequencing are indicated: A, AccI; B, BamHI; Bg, BglII;El, EcoRI; EV, EcoRV; H, HindIII; N, NcoI; P,. PstI; S, SmaI; Ss, SstI;X, XbaI.

FIGS. 14(a)-(c) depict the sequence of the human genomic P40 gene.Nucleotide numbering is relative to the initiator ATG. Amino acidsequences of the coding regions are given in three-letter code.Potential regulatory or signal sequences are underlined.

FIGS. 15(a)-(c) depict the sequences of the mouse genomic P40 gene.Nucleotide numbering is relative to the initiator ATG. Amino acidsequences of the coding regions are given in three-letter code.Potential regulatory or signal sequences are underlined.

FIG. 16 shows a comparison of the human and mouse P40 5'-(A) and 3'-untranslated regions (B). In (A), nucleotides are numbered 3' to 5'relative to the first nucleotide of the ATG start codon. Transcriptionstarts are underlined and conserved consensus motifs are boxed. In (B),nucleotides are numbered 5' to 3' starting with the first nucleotidefollowing the stop codon. Nucleotide sequences potentially involved inmRNA degradation are underlined and presumptive polyadenylation signalsare double-underlined.

FIG. 17 shows an autoradiograph illustrating the kinetics of P40 inactivated human peripheral blood mononuclear cells (PBMC). PBMC werestimulated with PMA and A23187, and RNA was extracted at the indicatedtimes. Northern blots were hybridized with a ³² P-labeled human P40 cRNAand exposed overnight.

FIG. 18 shows a dot blot illustrating the ability of various stimuli toinduce P40 in human PBMC. Unfractionated PBMC were stimulated asdescribed in Example 1. Three-fold dilutions of cytoplasmic RNA wereblotted on nitrocellulose and hybridized with ³² P-labeled P40 cDNA.

FIG. 19 illustrates the expression of P40 mRNA in CD4⁺ T cells. HumanPBMC were fractionated as described in Example 1 and stimulated for 24hours with PHA and PMA. Three-fold dilutions of cytoplasmic RNA wereblotted onto nitrocellulose and hybridized with ³² P-labeled P40 CDNA.RNA was prepared from 15 and 10×10⁶ cells in left and right panels,respectively.

FIG. 20 is a graphic illustration depicting the expression andbiological activity of recombinant human P40. The left panel showssurvival curves of a human T cell line (EL) incubated in medium withoutfactors (□), or in medium supplemented with saturating concentrations ofsemi-purified baculovirus-derived human P40 (), with a controlpreparation derived from cells infected with wild-type baculovirus (◯),with purified mouse recombinant P40 (▪) or with human IL-4 (▴). Allcultures were seeded with 50,000 cells on day 0. The right panel showsSDS-PAGE of ³⁵ S-labeled semi-purified recombinant human P40 (left lane)and of a control preparation (right lane).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a mammalian T cell growth factor whichcomprises a protein which supports, or is capable of supporting,interleukin 2 (IL2)-independent and interleukin 4 (IL4)-independentgrowth of helper T cells in the absence of antigen. In accordance withthe present invention and using the methods contained herein, said Tcell growth factor is biologically pure. By biologically pure is meant acomposition comprising said T cell growth factor. The composition maycomprise homogeneous T cell growth factor or may consist essentially ofT cell growth factor. As used in the specification and appended claims,supporting IL2-independent and IL4-independent growth of helper T cellsrefers to the ability for said cells to proliferate in the absence ofIL2 and/or IL4. This feature distinguishes the subject growth factorfrom others presently known. In accordance with the present invention,this ability is due to a novel and heretofore unknown T cell growthfactor. Hereinafter, said growth factor is referred to as P40. Asdefined herein, derivatives of P40 encompass synthetic and naturallyoccurring amino acid substitutions, deletions and/or insertions as willbe apparent to the skilled artisan. For example, non-essential aminoacid deletions, i.e., deletion of amino acids which do not affect theactivity of P40 are obtainable by genetic engineering means.

Furthermore, fragments of P40 are contemplated by the present invention.These fragments are peptides obtained from the P40 protein and may beprepared by proteolysis of purified P40. The peptides are purified byconventional means such as HPLC chromatography and the like, and areuseful in determining the P40 amino acid sequence, in preparingantibodies to specific domains of P40 and in identifying the P40 domainsinvolved in stimulating T cell growth.

An antigenic derivative of P40 is defined to be a portion of P40 whichis capable of reacting with an antibody specific to P40. All suchderivatives are encompassed by the subject invention.

Accordingly, P40 is a protein, and more particularly, a glycoprotein,capable of supporting long-term IL2-independent and IL4-independentgrowth of helper T cell lines in the absence of antigen, and is isolatedfrom helper T cell lines, especially mammalian lines like murine andhuman helper T cell lines. P40 is functionally distinct from all knowninterleukins and colony-stimulating factors. P40 is purified from thesupernatant (SN) of lectin-stimulated mouse helper T cell lines to aspecific activity of from about 10 U/mg to about 10¹⁰ U/mg, butgenerally to about 10⁸ U/mg and characterized as a basic (pI≈10 formurine P40) single chain protein with an Mr of from about 30 to about 40kDa.

P40 can be purified from the supernatant fluid of antigen stimulatedmouse helper T cell clones (TUC2.15 and TUC7.51). Briefly, thesupernatant fluid is concentrated and applied to a TSK-phenylchromatography column. Fractions with growth factor activity onfactor-dependent TS1 cells are pooled, further fractionated on a Mono-Qchromatography column, and the resulting active fractions applied to aC1 reversed-phase HPLC column. Pure murine P40 is eluted at aconcentration of about 35% acetonitrile.

Two observations indicate that P40 is a glycoprotein: (i) itsheterogeneous migration pattern in NaDodSO₄ /PAGE and (ii) its bindingto lentil lectin, which points to the presence of N-linked carbohydrateside chains. Consistent with this observation, a number of potentialN-glycosylation sites (Asn-X-Thr motif) have been identified in theprotein sequence. Moreover, additional evidence for extensiveglycosylation of the molecule is obtained in experiments withN-glycanase treatment, which reduced the Mr of P40 to about 15 kDa. P40is a stable molecule whose biological activity is not altered afterexposure to NaDodSO₄, acid pH or acetonitrile. By contrast, its activityis destroyed by 2-mercaptoethanol, which suggests that intramoleculardisulfide bridges play an important role in maintaining appropriatefolding of the molecule. P40 is also distinguished from known proteinson the basis of its complete amino acid sequence. The DNA and amino acidsequence of murine P40 and human P40 are described herein and indicatethat the two proteins are 55% homologous.

In addition to the aforementioned distinguishing structuralcharacteristics of P40, it also differs functionally from IL2. P40 iscompletely inactive on cytolytic T cell clones under conditions wheretheir response to IL2 is very strong; conversely, IL2 fails to supportlong-term antigen independent growth of helper T cell lines, whereas P40is very active in this system. To date, long-term growth of helper cellsin response to P40 means greater than two months and may be indefinite.In contrast with these differences, a correlation is observed betweenthe sensitivity of helper T cell lines to P40 and IL4, indicating that Tcell activation by these two molecules is similarly regulated. However,the range of activities of IL4, which also stimulates the growth of avariety of IL3-dependent cell lines and of cytolytic T cells (Mosmann etal., Proc. Natl. Acad. Sci. USA 83: 5654-5658, 1986; Widmer et al.,Nature 326: 795-798, 1987) is broader than that of P40, indicating thatthe functional overlap between the two factors, IL4 and P40, is onlypartial.

Another advantage of the subject T cell growth factor, P40, is thesurprising discovery that P40 is specific for helper T cell lines. Thisindicates the existence of a growth-stimulatory mechanism restricted tothe helper T cell subset. Such a mechanism is important for maintainingthe balance between the supply of helper T cell products like IL2 andIL4 and their increased consumption by other lymphocytes activated inthe course of the immune response.

While investigating the range of P40 activity, it was surprisinglydiscovered that P40 augments proliferation of IL3- or IL4-responsivecells in a synergistic manner. As used herein, IL3- and IL4-responsivecells are immune system cells which proliferate in response to IL3 orIL4, respectively. These cells may include IL3-dependent cells orIL4-dependent cells, but are not limited thereto. IL3-responsive cellsinclude helper T cells, stem cells, mast cells, eosinophils,neutrophils, monocytes, megakaryocytes, basophils, and erythropoidcells. IL4-responsive cells include helper T cells, activated cytotoxicT cells, macrophages, mast cells and B cells (Smith, K. A.,Biotechnology 7: 661-667, 1989).

Hence there is a strong synergism with respect to growth for cellsstimulated with P40 and IL3, or P40 and IL4. In a thymidine uptake assaywhich measures cellular proliferation, the combination of cytokines P40and IL3, or P40 and IL4, can stimulate thymidine uptake by a factorranging from about 4 to 40 above the stimulatory effect of any one ofthe cytokines. In general, the synergism between P40 and IL3, or P40 andIL4, is dose dependent and cell line dependent. For these proteins and agiven cell line, suboptimal doses of P40 range from about 1-25% ofoptimal P40 doses, suboptimal doses of IL4 range from about 5-30% ofoptimal IL4 doses, and close to optimal doese of IL3 range from about70-100% of optimal IL3 doses. This synergism provides a further methodto stimulate proliferation of IL3- and IL4-responsive cells, especiallyhelper T cells, and is therapeutically useful in treating immunedeficiencies, especially those diseases or disease states which benefitfrom proliferation of specific immune cells such as AIDS, or even fromgeneral proliferation of immune cells.

Further investigation of the biological properties of mammalian P40 showthat this cytokine has a broader spectrum of activities includingenhancement of cell survival, and may effect a wider variety of celltypes, generally of hemapoetic lineage. Human P40 prolonged survival ofhuman T cell lines maintained in culture in the presence of PHA,irradiated PBMC as feeders, and IL-4. Murine P40 also increased cellsurvival of mouse T cell lines, and in one instance, a human T cell line(see Example 15). Human P40 was apparently not active on mouseP40-dependent cell lines.

P40 does not appear to be constitutively expressed but can generally beinduced by T cell mitogens or with a combination of phorbal myristateacetate (PMA) and the calcium ionophore A23187. T cell mitogens whichinduced P40 in PBMC include phytohemagglutinin (PHA) and anti-CD3antibodies. Co-culturing PBMC with either of these mitogens and PMAfurther enhanced P40 expression, especially human P40. No inductionoccured when PBMC were treated with PMA alone, lipopolysaccharide orStaphylococcus aureus strain Cowan 1. In all cases expression wasmonitored by measuring the levels on P40 mRNA by dot blot analysis.

To investigate the pattern of P40 expression in human PBMC, cells werefractionated into resetting cells (representing primarily T cells) andnon-rosetting cells. The resetting cells were then further divided intoCD4⁺ and CD8⁺ T cells by fluorescence-activated cell sorting. Expressionof P40 was detected in resetting PBMC and CD4⁺ T cells, but not innon-rosetting PBMC or CD8⁺ T cells. These results indicate that P40 isprimarily expressed in helper T cells (CD4⁺ cells) in humans aspreviously found for murine P40.

It is within the scope of the present invention to include biologicallypure P40 in addition to homogenous and heterogenous compositionsthereof. Thus in accordance with the present invention, supernatant (SN)from a helper T cell line not requiring antigen or feeders comprisesP40. This SN is able to induce cell proliferation without furtherrequirement for antigen or feeder cells. As further described in Example1, the proliferation activity is not inhibited by either anti-IL4 oranti-IL2 receptor antibodies, indicating that said activity is mediatedneither directly nor indirectly by these molecules. The activeingredient in the aforementioned SN is shown to be, in accordance withthe present invention, P40. The SN is active on the test cells, TS1,inducing half-maximal proliferation at dilutions ranging of from about10⁻⁶ to about 10⁻² (v/v), and generally ranging from about 10⁻⁵ to about10⁻⁴ (v/v). Thus, in accordance with the present invention, the novel Tcell growth factor P40 is active in biologically pure form and inhomogenous and heterogeneous compositions. As exemplified herein, SNfluid is a form of heterogeneous composition of P40. Homogeneouscompositions are exemplified herein to include pharmaceuticalcompositions containing homogeneous preparations of P40, its activederivatives or fragments, and the like.

The T cell growth factor P40 is contemplated herein to be useful instimulating the proliferation of T helper cells in mammals. In apreferred embodiment, P40 is particularly useful in stimulating certainsubsets of T helper cells in mammals. Accordingly, P40 is a new anduseful therapeutic compound capable of stimulating specific cells withinthe immune cells. For example, this is particularly important for humanpatients carrying defects in certain subsets of T helper cells as may bethe case with various AIDS patients or immune compromised patients. Itshould also be noted that of the many advantages of the presentinvention, the proliferation of helper T cells by P40 will have theadditional effect of allowing increased amounts of other cytokines to beproduced. Accordingly, the present invention also contemplates a methodof treatment of immune deficiency compromising the administration of aproliferating effective amount of P40, an active derivative, or anactive fragment thereof, for a time and under conditions sufficient toeffect proliferation of helper T cells. In accordance with the presentinvention, the time required for the proliferation of helper T cellsranges from about two days to about seven days.

Accordingly, the subject invention contemplates a method for inducingand maintaining the proliferation of helper T cells, and preferable,certain subsets thereof, in a mammal which comprises administering tosaid mammal a proliferating-effective amount of a pharmaceuticalcomposition containing P40, an active derivative or fragment thereof,for a time and under conditions sufficient for said cells toproliferate. Additionally, a method for inducing and maintaining theproliferation of helper T cells, and preferably certain subsets thereof,in a mammal, is contemplated by this invention in which a nucleic acidmolecule encoding P40 is introduced into a T cell in such a manner thatsaid nucleic acid molecule is expressed intracellularly, butextrachromosomally of said cell or following integration into the genomeof said cell. In this case, the nucleic acid molecule is carried to saidT cell and transferred into said cell by a second nucleic acid molecule(e.g., various viruses). The first nucleic acid molecule is manipulatedsuch that it contains the appropriate signals for expression. That is,in accordance with the present invention, a method for proliferating Thelper cells in a mammal is contemplated comprising administering afirst nucleic acid molecule encoding P40, said nucleic acid moleculebeing contained in a pharmaceutically acceptable second nucleic acidcarrier molecule such that said first nucleic acid molecule enters a Tcell and is either maintained extrachromosomally or integrated into thegenome of said target all in such a manner that said first-nucleic acidmolecule is expressed so as to produce an effective amount of P40. Bynucleic acid molecule is meant the nucleotide sequence which encodes,directly or indirectly, P40 or a derivative thereof. A nucleic acidmolecule is defined herein to mean RNA or DNA.

The active ingredients of a pharmaceutical composition comprising P40are contemplated to exhibit excellent and effective therapeuticactivity, for example, in the treatment of immune compromised diseasesin mammals. Thus the active ingredients of the therapeutic compositionscomprising P40 exhibit helper T cell proliferative activity whenadministered in therapeutic amounts which depend on the particulardisease. For example, from about 0.5 ug to about 2000 mg per kilogram ofbody weight per day may be administered. The dosage regimen may beadjusted to provide the optimum therapeutic response. For example,several divided doses may be administered daily or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation. A decided practical advantage is that the active compound maybe administered in a convenient manner such as by the oral, intraveneous(where water soluble), intramuscular, subcutaneous, intranasal,intradermal or suppository routes. Depending on the route ofadministration, the active ingredients which comprise P40 may berequired to be coated in a material to protect said ingredients from theaction of enzymes, acids and other natural conditions which mayinactivate said ingredients. For example, the low lipophilicity of P40may allow it to be destroyed in the gastrointestinal tract by enzymescapable of cleaving peptide bonds and in the stomach by acid hydrolysis.In order to administer P40 by other than parenteral administration, P40should be coated by, or administered with, a material to prevent itsinactivation. For example, P40 may be administered in an adjuvant,co-administered with enzyme inhibitors or in liposomes. Adjuvantscontemplated herein include resorcinols, non-ionic surfactants such aspolyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzymeinhibitors include pancreatic trypsin inhibitor,diisopropylfluorophosphate (DFP) and trasylol. Liposomes includewater-in-oil-in-water P40 emulsions as well as conventional liposomes.

The active compounds may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof, and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases the form must be sterile and mustbe fluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, liquid polyethylene glycol, and the like), suitable mixturesthereof and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. The preventions of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

When P40 is suitably protected as described above, the active compoundmay be orally administered, for example, with an inert diluent or withan assimilable edible carrier, or it may be enclosed in hard or softshell gelatin capsule, or it may be compressed into tablets, or it maybe incorporated directly with the food of the diet. For oral therapeuticadministration, the active compound may be incorporated with excipientsand used in the form of ingestible tablets, buccal tablets, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. Suchcompositions and preparations should contain at least 1% of activecompound. The percentage of the compositions and preparations may, ofcourse, be varied and may conveniently be between about 5 to about 80%of the weight of the unit. The amount of active compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained. Preferred compositions or preparations according to thepresent invention are prepared so that an oral dosage unit form containsbetween about 10 ug and 1000 ug of active compound.

The tablets, troches, pills, capsules, and the like, may also containthe following: a binder such as gum gragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid, and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin may be added or a flavoring agent such aspeppermint, oil of wintergreen or cherry flavoring. When the dosage unitform is a capsule, it may contain, in addition to materials of the abovetype, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills or capsules may be coated with shellac,sugar or both. A syrup or elixir may contain the active compound,sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound may be incorporated intosustained-release preparations and formulations.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the novel dosageunit forms of the invention are dictated by and directly dependent on(a) the unique characteristics of the active material and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active material for the treatment ofdisease in living subjects having a diseased condition in which bodilyhealth is impaired as herein disclosed in detail.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit dosage form can, for example, contain the principalactive compound in amounts ranging from 0.5 ug to about 2000 mg.Expressed in proportions, the active compound is generally present infrom about 10 ug to about 2000 mg/ml of carrier. In the case ofcompositions containing supplementary active ingredients, the dosagesare determined by reference to the usual dose and manner ofadministration of the said ingredients.

As used herein "pharmaceutically acceptable carrier" includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and adsorption delaying agents, and the like. The useof such media agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, use thereof in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

A further aspect of this invention contemplates the use of P40 with IL3or IL4 in a method to stimulate proliferation of IL3- or IL4-responsivecells and in a method of treatment of immune deficiency. Such methodsare practiced in accordance with the therapeutic methods involving onlyP40 and as described herein. Likewise, pharmaceutical compositionscontaining P40 and IL3, or P40 and IL4 are provided in accordance withthose which contain P40 alone. Further in this regard, IL3 and IL4 arecommercially available and are used in therapeutically effectiveamounts. Pharmaceutically effective amounts of P40 when used inconjunction with IL3 or IL4 are the same as when P40 is used alone.Likewise, pharmaceutically effective amounts of IL3 or IL4 amounts canbe similar to those provided for P40 alone. Preferred compositions ofP40 and IL3 according to the present invention are prepared so that aunit dosage form contains each protein in an amount ranging from about0.5 ug to about 2000 mg. Preferred compositions of P40 and IL4 arelikewise prepared so that a unit dosage form contains each protein in anamount ranging from about 0.5 ug to about 2000 mg. In thesecompositions, the relative amount of P40 to IL3 or IL4 can be varied orthe same.

The present invention also relates to antibodies to P40, its derivativesor fragments. Such antibodies are contemplated to be useful indeveloping detection assays (immunoassays) for P40, especially duringthe monitoring of a therapeutic regimen and in the purification of P40.The antibodies may be monoclonal or polyclonal. Additionally, it iswithin the scope of this invention to include any second antibodies(monoclonal or polyclonal) directed to the first antibodies discussedabove. The present invention further contemplates use of these secondantibodies in detection assays and, for example, in monitoring theeffect of an administered pharmaceutical preparation. Furthermore, it iswithin the scope of the present invention to include antibodies to theglycosylated regions of P40, and to any molecules complexed with saidP40. Accordingly, in accordance with this invention, an antibody to P40encompasses antibodies to P40, or antigenic parts thereof, and to anyassociated molecules (e.g., glycosylated regions, lipid regions, carriermolecules, and the like).

The P40, or parts thereof, considered herein are purified, asexemplified in Example 3, then utilized in antibody production. Bothpolyclonal and monoclonal antibodies are obtainable by immunization withP40, its derivatives, polypeptides or fragments, and either type ofantibody is utilizable for immunoassays. The methods for obtaining bothtypes of sera are well known in the art. Polyclonal sera are lesspreferred, but are relatively easily prepared by injection of a suitablelaboratory animal with an effective amount of the purified P40, or partsthereof, collecting serum from the animal and isolating specific sera byany of the known immunoadsorbent techniques. Although antibodiesproduced by this method are utilizable in virtually any type ofimmunoassay, they are generally less favored because of the potentialheterogeneity of the product.

The use of monoclonal antibodies in the present immunoassay isparticularly preferred because of the ability to produce them in largequantities and the homogeniety of the product. The preparation ofhybridoma cell lines for monoclonal antibody production derived byfusing an immortal cell line and lymphocytes sensitized against theimmunogenic preparation can be done by techniques which are well knownto those who are skilled in the art. (See, for example, Douillard, J. Y.and Hoffman, T., "Basic Facts About Hybridomas", in Compendium ofImmunology, Vol. II, L. Schwartz (Ed.) (1981); Kohler, G. and Milstein,C., Nature 256: 495-497 (1975); European Journal of Immunology 6:511-519 (1976); Koprowski et al., U.S. Pat. No. 4,172,124, Koprowski etal., U.S. Pat. No. 4,196,265 and Wands, U.S. Pat. No. 4,271,145, theteachings of which are herein incorporated by reference.

Unlike preparation of polyclonal sera, the choice of animal formonoclonal antibody production is dependent on the availability ofappropriate immortal lines capable of fusing with lymphocytes thereof.Mouse and rat have been the animals of choice in hybridoma technologyand are preferably used. Humans can also be utilized as sources forsensitized lymphocytes if appropriate immortalized human (or nonhuman)cell lines are available. For the purpose of the present invention, theanimal of choice may be injected with from about 1 mg to about 20 mg ofthe purified P40 or parts thereof. Usually the injecting material isemulsified in Freund's complete adjuvant. Boosting injections may alsobe required. The detection of antibody production can be carried out bytesting the antisera with appropriately labeled antigen. Lymphocytes canbe obtained by removing the spleen or lymph nodes of sensitized animalsin a sterile fashion and carrying out fusion. Alternatively, lymphocytescan be stimulated or immunized in vitro, as described, for example, inC. Reading, J. Immunol. Meth. 53: 261-269, 1982.

A number of cell lines suitable for fusion have been developed, and thechoice of any particular line for hybridization protocols is directed byany one of a number of criteria such as speed, uniformity of growthcharacteristics, deficiency of its metabolism for a component of thegrowth medium, and potential for good fusion frequency.

Intraspecies hybrids, particularly between like strains, work betterthan interspecies fusions. Several cell lines are available, includingmutants selected for the loss of ability to secrete myelomaimmunoglobulin. Included amont these are the following mouse myelomalines: MPC₁₁ -X45-6TG, P3-NS1-1-Ag4-1, P3-X63-Ag8, or mutants thereofsuch as X63-Ag8.653, SP2-0-Ag14 (all BALB/C derived), Y3-'Ag1.2.3 (rat)and U266 (human).

Cell fusion can be induced either by virus, such as Epstein-Barr orSendai virus, or polyethylene glycol. Polyethylene glycol (PEG) is themost efficacious agent for the fusion of mammalian somatic cells. PEGitself may be toxic for cells, and various concentrations should betested for effects on viability before attempting fusion. The molecularweight range of PEG may be varied from 1000 to 6000. It gives bestresults when diluted to about 20% to about 70% (w/w) in saline orserum-free medium. Exposure to PEG at 37° for about 30 seconds ispreferred in the present case, utilizing murine cells. Extremes oftemperature (i.e., about 45° C.) are avoided, and preincubation of eachcomponent of the fusion system at 37° C. prior to fusion gives optimumresults. The ratio between lymphocytes and malignant cells is optimizedto avoid cell fusion among spleen cells and a range of from about 1:1 toabout 1:10 gives good results.

The successfully fused cells can be separated from the myeloma line byany technique known by the art. The most common and preferred method isto choose a malignant line which is Hypoxanthine Guanine PhosphoribosylTransferase (HGPRT) deficient, which will not grow in anaminopterin-containing medium used to allow only growth of hybrids andwhich is generally composed of hypoxanthine 1×10⁻⁴ M, aminopterin 1×10⁻⁵M and thymidine 3×10⁻⁵ M, commonly known as the HAT medium. The fusionmixture can be grown in the HAT-containing culture medium immediatelyafter the fusion 24 hours later. The feeding schedules usually entailmaintenance in HAT medium for two weeks and then feeding with eitherregular culture medium or hypoxanthine, thymidine-containing medium.

The growing colonies are then tested for the presence of antibodies thatrecognize the antigenic preparation. Detection of hybridoma antibodiescan be performed using an assay where the antigen is bound to a solidsupport and allowed to react to hybridoma supernatants containingputative antibodies. The presence of antibodies may be detected by"sandwich" techniques using a variety of indicators. Most of the commonmethods are sufficiently sensitive for use in the range of antibodyconcentrations secreted during hybrid growth.

Cloning of hybrids can be carried out after 21-23 days of cell growth inselected medium. Cloning can be performed by cell limiting dilution influid phase or by directly selecting single cells growing in semi-solidagarose. For limiting dilution, cell suspensions are diluted serially toyield a statistical probability of having only one cell per well. Forthe agarose technique, hybrids are seeded in a semi-solid upper layer,over a lower layer containing feeder cells. The colonies from the upperlayer may be picked up and eventually transferred to wells.

Antibody-secreting hybrids can be grown in various tissue cultureflasks, yielding supernatants with variable concentrations ofantibodies. In order to obtain higher concentrations, hybrids may betransferred into animals to obtain inflammatory ascites.Antibody-containing ascites can be harvested 8-12 days afterintraperitoneal injection. The ascites contain a higher concentration ofantibodies but include both monoclonals and immunoglobulins from theinflammatory ascites. Antibody purification may then be achieved by, forexample, affinity chromatography.

The presence of P40 contemplated herein, or antibodies specific forsame, in a patients serum, tissue or tissue extract, can be detectedutilizing antibodies prepared as above, either monoclonal or polyclonal,in virtually any type of immunoassay. A wide range of immunoassaytechniques are available as can be seen by reference to U.S. Pat. Nos.4,016,043; 4,424,279 and 4,018,653. This, of course, includes bothsingle-site and two-site, or "sandwich", assays of the non-competitivetypes, as well as in the traditional competitive binding assays.Sandwich assays are among the most useful and commonly used assays andare favored for use in the present invention. A number of variations ofthe sandwich assay technique exist, and all are intended to beencompassed by the present invention. Briefly, in a typical forwardassay, an unlabeled antibody is immobilized in a solid substrate and thesample to be tested brought into contact with the bound molecule. Aftera suitable period of incubation, for a period of time sufficient toallow formation of an antibody-antigen secondary complex, a secondantibody, labeled with a reporter molecule capable of producing adetectable signal is then added and incubated, allowing time sufficientfor the formation of a tertiary complex of antibody-antigen-labeledantibody (e.g., antibody-P40-antibody). Any unreacted material is washedaway, and the presence of the antigen is determined by observation of asignal produced by the reporter molecule. The results may either bequalitative, by simple observation of the visible signal, or may bequantitated by comparing with a control sample containing known amountsof hapten. Variations on the forward assay include a simultaneous assay,in which both sample and labeled antibody are added simultaneously tothe bound antibody, or a reverse assay in which the labeled antibody andsample to be tested are first combined, incubated and then added to theunlabeled surface bound antibody. These techniques are well known tothose skilled in the art, and the possibility of minor variations willbe readily apparent.

In the typical forward sandwich assay, a first antibody havingspecificity for P40, or antigenic parts thereof, contemplated in thisinvention, is either covalently or passively bound to a solid surface.The solid surface is typically glass or a polymer, the most commonlyused polymers being cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride or polypropylene. The solid supports may be in theform of tubes, beads, discs or microplates, or any other surfacesuitable for conducting an immunoassay. The binding processes arewell-known in the art and generally consist of cross-linking, covalentlybinding or physically absorbing the molecule to the insoluble carrier.Following binding, the polymer-antibody complex is washed in preparationfor the test sample. An aliquot of the sample to be tested is then addedto the solid phase complex and incubated at 25° C. for a period of timesufficient to allow binding of any subunit present in the antibody. Theincubation period will vary but will generally be in the range of about2-40 minutes. Following the incubation period, the antibody subunitsolid phase is washed and dried and incubated with a second antibodyspecific for a portion of the hapten. The second antibody is linked to areporter molecule which is used to indicate the binding of the secondantibody to the hapten. By "reporter molecule," as used in the presentspecification, is meant a molecule which, by its chemical nature,provides an analytically identifiable signal which allows the detectionof antigen-bound antibody. Detection may be either qualitative orquantitative. The most commonly used reporter molecules in this type ofassay are either enzymes, fluorophores or radionuclide containingmolecules (i.e., radioisotopes). In the case of an enzyme immunoassay,an enzyme is conjugated to the second antibody, generally by means ofglutaraldehyde or periodate. As will be readily recognized, however, awide variety of different conjugation techniques exist, which arereadily available to the skilled artisan. Commonly used enzymes includehorseradish peroxidase, glucose oxidase, β-galactosidase and alkalinephosphatase, among others. The substrates to be used with the specificenzymes are generally chosen for the production, upon hydrolysis by thecorresponding enzyme, of a detectable color change. For example,p-nitrophenyl phosphate is suitable for the use with alkalinephosphatase conjugates; for peroxidase conjugates, 1,2-phenylenediamine,5-aminosalicyclic acid, or tolidine, are commonly used. It is alsopossible to employ fluorogenic substrates, which yield a fluorescentproduct rather than the chromogenic substrates noted above. In allcases, the enzyme-labeled antibody is added to the first antibody haptencomplex, allowed to bind, and then to the first antibody hapten complex,allowed to bind, and then the excess reagent is washed away. A solutioncontaining the appropriate substrate is then added to the ternarycomplex of antibody-antigen-antibody. The substrate will react with theenzyme linked to the second antibody, giving a qualitative visualsignal, which may be further quantitated, usuallyspectrophotometrically, to give an indication of the amount of haptenwhich was present in the sample.

Alternatively, fluorescent compounds, such as fluorescein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labeled antibody adsorbs the light energy,inducing a state of excitability in the molecule, followed by emissionof the light at a characteristic color visually detectable with a lightmicroscope. As in the EIA, the fluorescent labeled antibody is allowedto bind to the first antibody-hapten complex. After washing off theunbound reagent, the remaining ternary complex is then exposed to thelight of the appropriate wavelength, the fluorescence observed indicatesthe presence of the hapten of interest. Immunofluorescence and EIAtechniques are both very well established in the art and areparticularly preferred for the present method. However, other reportermolecules, such as radioisotope, chemiluminescent or bioluminsecentmolecules, may also be employed. It will be readily apparent to theskilled technician how to vary the procedure to suit the requiredpurpose. It will also be apparent that the foregoing can be used todetect directly or indirectly (i.e., via antibodies) the P40 of thisinvention.

Accordingly, the present invention is also directed to a kit for therapid and convenient assay of P40 in mammalian body fluids (e.g. serum,tissue extracts, tissue fluids), in vitro cell culture supernatants, andcell lysates. The kit is compartmentalized to receive a first containeradapted to contain an antibody to P40, or to an antigenic componentthereof, and a second container adapted to contain a second antibody toP40, or to an antigenic component thereof, said second antibody beinglabeled with a reporter molecule capable of giving a detectable signalas hereinbefore described. If the reporter molecule is an enzyme, then athird container adapted to contain a substrate for said enzyme isprovided. In an exemplified use of the subject kit, a sample to betested for P40 is contacted with the contents of the first container fora time and under conditions for P40, if present, to bind to theantibodies contained in said first container. After removal of unboundmaterial (e.g. by washing with sterile phosphate buffered saline), thesecondary complex is contacted with the contents of the secondcontainer. If the antibodies of the first container have bound to P40,then the antibodies of the second container bind to the secondarycomplex to form a tertiary complex and, since said second antibodies arelabeled with a reporter molecule, when subjected to a detecting means,the tertiary complex is detected.

Another aspect of this invention relates to a recombinant nucleic acidor an isolated nucleic acid molecule, said molecule defined herein to beDNA or RNA, encoding P40 or parts thereof. In one embodiment therecombinant nucleic acid molecule is complementary DNA (cDNA). It isconsidered within the scope of the present invention to include the cDNAmolecule encoding mammalian P40, preferable murine and human P40, or toregions or parts thereof including any base deletion, insertion orsubstitution or any other alteration with respect to nucleotide sequenceor chemical composition (e.g. methylation and glycosylation).Additionally, the present invention is directed to restriction fragmentsand synthetic fragments from a nucleic acid encoding mammalian P40. P40encoded by cDNA or a recombinant DNA is referred to herein asrecombinant P40. Moreover, another embodiment of this invention isdirected to the genomic P40 gene, which may include recombinant cloneslike cosmids encoding the entire gene or subclones encoding exons,introns or any region of the mammalian P40 gene. Recombinant DNAencoding such subregions of the gene are useful as hybridization probesto detect the presence of P40 genes.

Methods considered useful in obtaining recombinant P40 cDNA arecontained in Maniatis et al., 1982, in Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York, pp. 1-545, for example,or any of the myriads of laboratory manuals on recombinant DNAtechnology which are widely available. Briefly, polyadenylated mRNA isobtained from stimulated helper T cells and fractionated on agarosegels. Optionally, aliquots of mRNA can be injected into Xenopus laevisoocytes for translation and assayed for P40 activity using the methodscontained herein to enriched fractions of MRNA translating into P40active molecules. Alternatively, mRNA not enriched is used as templatefor cDNA synthesis. Libraries of CDNA clones are constructed in the Pst1site of the vector pBR322 (using homopolymer tailing) or in a variety ofother vectors (e.g. the Okayama-Berg cDNA cloning vectors, Messing cDNAcloning vectors and the like). Specific cDNA molecules in a vector insaid library are then selected by using specific oligonucleotidesdesigned, based on amino acid sequences contained within P40, to encodeat least part of said sequence. Particularly useful is the internal,partial amino acid sequence of murine P40 obtained after cyanogenbromide treatment which comprises:

    NH.sub.2 -Ala Gly Asn Thr Leu Ser Phe Leu Lys Ser Leu Leu Gly Thr Phe Gln Lys Thr Glu.

Olignucleotide sequences based on the foregoing amino acid sequence areparticularly useful in identifying cDNA clones encoding P40 or itsderivatives. Thus, poly(A)⁺ RNA can be prepared from the murine helper Tcell line TUC7.51 after 24 hours stimulation with Concanavalin A (Con A)and used as a template for cDNA synthesis. The cDNA can be cloned intoBamHI site of a pUC8 vector, transformed into E. coli and screened usinga 64-fold degenerate probe corresponding to the amino acid sequenceFQKTEMQ and subsequently with a 128-fold degenerate probe correspondingto the amino acid sequence ENLKDDP (See Example 9 for the exact sequenceof the probes). The resulting positive clones are useful to isolateother mammalian genomic P40 genes and cDNAs. For example, the murinecDNA clone is used to screen a human genomic library, or other mammaliangenomic library, to identify either the entire genomic gene or at leastan exon thereof. If only a portion of the gene is isolated by thismethod, the remainder of the gene can be isolated by "chromosomalwalking" with the new clone. Further, a genomic clone is particularlyuseful to isolate a cDNA clone and vice versa, especially from the samespecies. Thus, the murine cDNA clones can be used to isolate the murinegenomic P40 gene.

The cDNA sequence encoding murine P40 is set forth below with thecorresponding amino acid sequence: ##STR1##

The cDNA sequence with the corresponding amino acid sequence of humanP40 is set forth below: ##STR2##

Once identified, cDNAs or recombinant DNAs encoding all or part ofrecombinant P40 are ligated into expression vectors. Additional geneticmanipulation is routinely carried out to maximize expression of the cDNAin the particular host employed. Accordingly, P40 may be synthesized invitro by inserting said cDNA sequence into a replicable expressionvector, transforming the resulting recombinant molecule into a suitablehost and then culturing or growing the transformed host under conditionsrequisite for the synthesis of the molecule. The recombinant moleculedefined herein should comprise a nucleic acid sequence encoding adesired polypeptide inserted downstream of a promoter, a eukaryotic orprokaryotic replicon and a selectable marker such as resistance to anantibiotic.

A promoter consists of a specific nucleic acid sequence that is operablylinked to the DNA encoding the desired polypeptide which is capable ofeffecting expression of said polypeptide. Likewise, the promoter can bereplaced or augmented by any other genetic elements capable of effectinggene expression, including such elements as enhancers, transcriptionterminators, poly(A) signals and the like. The latter three elements arenot always necessary and their use will depend on both the vector andhost system used for gene expression. The need for any of these elementscan be easily determined by one skilled in the art. Promoters are DNAsequence elements for controlling gene expression, in particular, theyspecify transcription initiation sites. Prokaryotic promoters that areuseful include the lac promoter, the trp promoter, the P_(L) and P_(R)promoters of lambda and the T7 polymerase promoter. Eukaryotic promotersare especially useful in the invention and include promoters of viralorigin, such as the SV40 late promoter and the Molony Leukemia VirusLTR, yeast promoters and any promoters or variations of promotersdesigned to control gene expression, including genetically-engineeredpromoters. Control of gene expression includes the ability to regulate agene both positively and negatively (i.e., turning gene expression on oroff) to obtain the desired level of expression.

One skilled in the art has available many choices or replicableexpression vector, compatible hosts and well-known methods for makingand using the vectors. Recombinant DNA methods are found in any of themyriad of standard laboratory manuals on genetic engineering.

The recombinant molecule may also require a signal sequence tofacilitate transport of the synthesized polypeptide to the extracellularenvironment. Alternatively, the polypeptide may be retrieved by firstlysing the host cell by a variety of techniques such as sonication,pressure dissintegration or toluene treatment. Hosts contemplated inaccordance with the present ivention can be selected from the groupcomprising prokaryotes (e.g., Escherichia coli, Bacillus, sp.,Pseudomonas sp.) and eukaryotes (e.g., mammalian cells, yeast and fungalcultures, insect cells and plant cultures). The artisan will alsorecognize that a given amino acid sequence can undergo deletions,substitutions and additions of nucleotides or triplet nucleotides(codons). Such variations are all considered within the scope of thepresent invention and may be prepared by site-directed mutagenesistechniques. Additionally, depending on the host expression recombinantP40, said P40 may or may not be glycosylated. Generally, eukaryoticcells, for example mammalian T cells and the like, provide glycosylated,recombinant P40. Prokaryotic cells, for example bacteria such asEscherichia coli and the like, do not glycosylate proteins. Hence, bothglycosylated and nonglycoslated recombinant P40 are encompassed by thepresent invention.

Yet another aspect of the present invention provides transformantmicrooganisms and cultured cells containing the instantexpression-vectors. Transformant microorganisms and cultured cells aremade by introducing the replicable expression vector encoding mammalianP40, a derivative or a fragment thereof, into the desired cell ormicroorganisms by transformation, transfection or infection or virus orbacteriophage particles. Processes for transformation are well-known inthe art and include, but are not limited to CaCl₂ treatment andelectroporation for bacterial cells and CaPO₄ co-precipitation,protoplast fusion and electroporation for eukaryotic cells. Directinfection can be used when the vectors are viruses or bacteriophages.The detailed methods for these techniques can be found in standardlaboratory manuals on recombinant DNA technology. The invention furthercontemplates any method for incorporating DNA into a host organism.

Another aspect of the present invention relates to the helper T celllines which produce P40. As defined herein, P40 or compositionscomprising same, stimulate the development of permanentantigen-independent T helper cell lines which are maintained bysubcultivation every 3 to 4 days in medium with P40. Even moreparticularly, the present invention is directed to TS1, one of thefactor-dependent cell lines derived from TUC2.15.

The following examples further illustrate the present invention.

EXAMPLE 1 Materials and Methods

Medium

Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetalbovine serum (FCS), 50 uM β-mercaptoethanol, 0.55 mM L-arginine, 0.24 mML-asparagine and 1.25 mM L-glutamine are used for most cell lines exceptfor 7TD1 and BCL1 which are grown in Iscove's medium.

T Cell Clones and Lines

Helper T cell lines are established and maintained in the absence ofexogenous growth factors as described by Van Snick et al., Proc. Natl.Acad. Sci. USA 83:9679-9683, 1986. Lines TUC2 and TUC7 are derived fromC57BL/6 mice immunized with keyhole limpet hemocyanin. Line TUC5 isobtained from the same strain of mice but after immunization with humantransferrin. TUC13 is an allospecific BALB/c anti-C57B/6 line.Individual clones are derived from these lines by limiting dilution inthe presence of 10% (v/v) medium conditioned by rat spleen cellsstimulated with concanavalin A, and are denoted TUCx.y (where x standsfor the number of the line and y for the number of the clone). Theseclones are subsequently expanded and maintained without exogenous growthfactors like the parental cell lines. Cytolytic T cell clones of DBA/2origin directed against syngeneic P815 mastocytoma are maintained with50% (v/v) mixed lymphocyte culture medium as described by Maryanski etal., Eur. J. Immunol. 12:401-406, 1982. For use in growth factor assays,the T cells are separated from feeder cells by centrifugation over alayer of Lymphoprep (Nycomed AS, Oslo, Norway) washed and incubated at5×10⁴ cells/well. Proliferations are measured on day 3 after a 6 hrpulse with methyl-labeled ³ H!-thymidine (0.5 uCi/well).

Preparation of Helper T Cell Supernatants

TUC2.15 and TUC7.51 cells, obtained from cultures stimulated 2 weeksearlier with antigen and feeder cells, are adjusted to 2×10⁶ cells/mland incubated for 2-3 days in medium containing 0.5% (v/v) FCS andconcanavalin A (ConA, 5 ug/ml). Supernatants (SN) are collected bycentrifugation at 10,000 g for 20 min. When used for culture, crude SNare supplemented with 0.1M methyl-α-D-mannoside.

TS1 Growth Factor Assay

Factor-dependent TS1 cells are cultured in 1% (v/v) TUC2.15 SN. Beforeuse in the growth factor assay, the cells are washed free of SN andcultured at a density of 3×10³ cells/well in 200 ul with serialdilutions of samples to be tested. After 3 days, cell growth is measuredby colorimetric determination of hexosaminidase levels according toLandegren, J. Immunol. Methods 67:379-388, 1984. The dilution givinghalf-maximal absorbance at 405 nm is arbitrarily assigned one U/ml ofactivity.

Other Cell Lines

CTLL-2 (Gillis et al., J. Immunol. 120:2027-2032, 1978) is grown with100 U/ml of human recombinant IL-2 DA-1 (Ihle et al., Adv. Viral Oncol.4:95-137, 1984), Ea3.15 (Palacios et al., J. Exp. Med. 152:1036-1047,1980) with 10% (v/v) WEHI-3 SN as a source of IL3 and 7TD1 with a 1/500dilution of TUC2.15 SN as a source of IL6 (Van Snick et al. supra).Assays using these cell lines are carried out as described for the TS1line and proliferations are measured either by hexosaminidasedeterminations or by thymidine incorporation. In vivo passaged BCLIcells (Slavin et al., Nature 272:624-626, 1978) are frozen in aliquotsand thawed just before use. Proliferation of BCL1 is measured bythymidine incorporation in 7 day-old cultures seeded with 10⁴cells/well.

Cytokines and Growth Factors

Purified natural human ILLβ (Van Damme et al., Nature 314:266-268,1985), recombinant human IL2 (Devos et al., Nucleic Acids Res.11:4307-4323) and purified murine IL3 (Ihle et al., J. Immunol.129:2431-2436, 1982) are as described. Human recombinant granulocytecolony-stimulating factor (G-CSF) and mouse recombinantgranulocyte-macrophage colony stimulating factor (GM-CSF) is describedby DeLamarter et al., EMBO J. 4:2575-2581, 1985. Platelet-derived growthfactor (PDGF) is described by Heldin et al., Proc. Natl. Acad. Sci. USA76:3722-3726, 1979. Epidermal growth factor (EGF) is purchased fromBoehringer Mannheim (Fed. Rep. Germany). Mouse IL4, IL5 and IL6 arepurified as described by Van Snick, supra, and Vink et al., Eur. J.Immunol. 18: 607-612, 1988.

Antibodies

Anti-IL4 antibody 11B11 (Ohara et al., Nature 315:333-336, 1985) andanti-IL2 receptor antibody 5A2 (Moreau et al., Eur. J. Immunol.17:929-935, 1987) are as described.

Purification of TS1 Growth Factor

Adsorption to silicic acid and gel filtration is performed as described(Van Snick supra). Active fractions from the gel filtration column arepooled, concentrated by ultrafiltration on an Amicon YM-10 membrane inthe presence of 10⁻⁴ (v/v) dilution of Tween 20 and transferred to 1MNa₂ SO₄ buffered to pH 7.0 with 0.1M sodium phosphate before injectiononto a TSK-Phenyl column (LKB, Bromma, Sweden) equilibrated in the samebuffer. After a 10 min wash in the starting buffer, elution is carriedout at 0.6 ml/min with a linear gradient of a 1:1 mixture of a sodiumphosphate buffer (0.1M, pH 7.0) and ethylene glycol. Active fractionsare further fractionated on a MonoQ column (Pharmacia Fine Chemicals,Uppsala, Sweden) equilibrated in 20 mM ethanolamine-HCl pH 9.5, 20 mMNaCl and 10⁻⁴ (v/v) Tween 20. The column is developed at 0.8 ml/min witha 30 min linear gradient of NaCl (8 mM/min). Pooled active fractions areconcentrated and adjusted to contain 0.05% (w/v) trifluoroacetic acid(TFA) before injection on a C1 25-nm pore-size TSK TMS-250 HPLC column(LKB). The column is developed for the first 10 min with a lineargradient from 0 to 35% (w/v) acetonitrile in 0.05% (w/v) TFA, which isfollowed by a shallow 35-36% gradient for the next 60 min. Flow rate isadjusted to 0.8 ml/min; 1 min fractions are collected in Eppendorf tubescontaining 10 ul of 1M NH₄ HCO₃ and 5 ul of Tween 20 (1% (v/v) in water)and lyophilised. Total protein is measured fluorometrically withbenzoxanthene following Neuhoff et al., Hoppe-Seyler's Z. Physiol. Chem.360:1657-1670, 1979. The purity of the final product is assessed byNaDodSO₄ /PAGE in 12% (w/v) acrylamide gels. Isoelectric focusing isperformed with a LKB (Bromma, Sweden) vertical gel apparatus. Materialis recovered from gels by overnight incubation in 130 mM NaCl containingTween 20 (10⁻⁴ v/v) and 10 mM sodium phosphate, pH7.0. Affinitychromatography on lentil lectin-Sepharose is done following he proceduredescribed by the manufacturer (Pharmacia, Uppsala, Sweden).

Amino Acid Sequence Analysis

Automated amino acid sequence analysis is performed with an AppliedBiosystems sequencer (Model 477A) equipped with an on-linephenylthiohydanthoin amino acid analyzer (Model 120A). In situ cyanogenbromide cleavage of P40 (≈10 ug) is performed on the glass fiber sampledisk of the gas-phase sequencer according to a procedure described bySimpson et al. Biochem. Internat. 8:787-791, 1984, hereinafter SimpsonI. Sequence comparisons are made with the following databases: ProteinSequence database of PIR, National Biomedical Research Foundation(release 15.0, December 1987); Swiss-Prot Protein Sequence Data Bankversion 5 (September 1987, compiled by A. Bairoch, University of Geneva,Medical Biochemistry Department, 1211 Geneva 4, Switzerland); G. B.trans Protein Data Base Release 1.0 (August 1987) compiled from GENBANKrelease 50.0 by J. Coventry, Walter and Eliza Hall Institute of MedicalResearch, Parkville 3050 Australia; and PG trans Protein Data Baserelease 38.0 (December 1985) GENBANK, Instit. Pasteur, Paris, France.

S1 Nuclease Protection Assay

The transcription sites of the human and murine P40 genes weredetermined by S1 nuclease mapping as described in Davis L. G. et al.(1988) Methods in molecular biology. Elsevier Science Publishing Co.Inc., New York. For the murine gene, a single-stranded probe, including163 nucleotides, downstream from the initiator ATG and 421 bases of the5' flanking region, was constructed using a 584 bp EcoRV/BanII fragmentcloned in M13. Similarly, a human P40 probe was constructed using a 345bp BglII/BamHI fragment containing the first 77 bp of the codingsequence and 268 bp of the 5' flanking region of the human P40 gene.These probes were hybridized with poly(A)⁺ RNA from a ConA-activatedmouse helper T cell clone (TUC7.51) and from PHA-PMA stimulated humanPBMC, respectively.

Southern blotting

Mouse high molecular weight DNA was isolated from T cell leukemia L1210and from mastocytoma P815. Human DNA was from lymphoblastoid cell lineCESS and from HeLa cells. DNAs were digested with restrictionendonucleases and electrophoresed on a 0.8% agarose gel. Southern blotswere hybridized with ³² P-labeled mouse (clone P40.2B4) or human (clonecH40.4) P40 cDNA.

Cellular preparations

Peripheral blood mononuclear cells (PBMC) were prepared by Lymphoprep(Nycomed AS, Oslo, Norway) density gradient centrifugation. T cells wereseparated by rosetting, using sheep red blood cells treated withaminoethylisothiouronium bromide (Sigma, St. Louis, Mo.). This procedureyielded populations that were 90% CD3⁺ as determined by flow cytometry.In some experiments, T cells were further enriched by nylon woolfiltration prior to sorting of CD4⁺ and CD8⁺ cells by flow cytometry.The resulting preparations contained 92-94% CD4⁺ or CD8⁺ cells.Stimulations were performed for 24 hours in RPMI 1640 mediumsupplemented with 10% fetal calf serum. Phytohemagglutinin-P (DifcoLaboratories, Inc., Detroit, Mich.) was added at 30 μg/ml, PMA at 3ng/ml and ionophore A23187 at 10 ng/ml. Anti-CD3 antibody (OKT3) wasused at 10 μg/ml, LPS (E. coli 055:B5, Difco) at 20 μg/ml andStaphylococcus aureus strain Cowan 1 (Calbiochem) at 10 μg/ml.

RNA analysis

Northern blots were prepared on nylon filters with 10 μg total RNAisolated as described Glisin, V. et al. (1974) Biochemistry 13:2633! andfractionated by electrophoresis in 1.2% agarose gels with 6%formaldehyde. Gels were stained with ethidium bromide prior to transferand the RNA content of each lane was verified after transfer byexamining the filters under U.V. light. Hybridizations were carried outwith ³² P-labeled cRNA probes essentially as described in Zinn, K. etal. (1983) Cell 34:865. Cytoplasmic RNA was extracted according toManiatis et al. (1982), and denatured with formaldehyde before blottingonto nitrocellulose filters. Hybridizations were performed as describedVan Snick, J. et al. (1988) Eur. J. Immunol. 18:193! with a ³² P-labeledhuman P40 cDNA and with a chicken β-actin probe as a control.

The exact methodology of amino acid sequence determination is describedbelow:

A. Materials: Tween 20, guanadine hydrochloride (Sequenal grade) andtrifluoroacetic acid (F₃ ACOH; 99+% pure grade) were purchased fromPierce Chemical Co. (Rockford, Ill., USA). Iodoacetic acid (purissgrade) was obtained from Fluka (Buchs, Switzerland) and wasrecrystallized prior to use. Dithiothreitol was from Calbiochem (LaJolla, Calif., USA). Sodium chloride (Aristar grade) and aceticanhydride were purchased from BDH (Poole, UK). Cyanogen bromide (Univargrade) was from Ajax Chemical Co. (Sydney, Australia). All otherchemicals were of the highest grade commercially available.

Trypsin (treated with tosylphenylethylchloromethane) and chymotrypsinwere purchased from Worthington Biochemical Co. (New Jersey, USA).Staphylococcus aureus strain V8 protease was obtained from Miles Co.(Napperville, Ill., USA). Endoproteinase AspN from a Pseudomonas fragimutant and N-glycanase F were obtained from Boehringer Mannheim GmbH(West Germany). All organic solvents were HPLC grade (Chromar grade,Mallinckrodt, Ky., USA). Deionized water, obtained from a tandemMilli-RO and Milli-Q system (Millipore, Inc., Massachusetts, USA) wasused for all buffers.

B. Preparation of murine S-carboxymethyl-P40 (Cm-P40): P40 (15 ug in 120ul 35% aqueous acetonitrile containing 0.1% (v/v) F₃ AcOH and 0.02%Tween 20 was concentrated to approximately 10 ul by centrifugalcentrifugation (Savant Ind. Hicksville, N.Y.), diluted to 160 ul with7.5M guanidine.HCL containing 0.2M Tris.HCl buffer, pH 8.5, 0.002M EDTAand 0.02% (v/v) Tween 20 and then reduced with dithiotreitol (0.015M) at40° C. for 4.5 h. Alkylation was to very small concentrations of SN,half-maximal proliferation being obtained at dilutions between 10⁻⁵ and10⁻⁴ (v/v). To determine the specificity of the TS1 assay, cells areincubated with a variety of purified growth factors or crude SN andfound that only IL4 and TUC2.15 SN support TS1 growth (Table 2). Sinceanti-IL4 antibodies fail to inhibit the effects of TUC2.15 SN, theaforementioned activity is a new T cell growth factor.

                  TABLE 1                                                         ______________________________________                                        Proliferation of TUC2.15 Helper T Cells Induced                               by Autologous Supernatant (SN); Independence from                             IL2 and IL4                                                                             Proliferation in response to                                        Antibodies Added                                                                          IL2        IL4     TUC2.15 SN                                     ______________________________________                                                               (kepm)                                                 none        152        18      37                                             anti-IL2 receptor                                                                          4         16      32                                             anti-IL4    156         1      33                                             ______________________________________                                    

TUC2.15 helper T cells (5×10⁴ /well) are incubated for 3 days with IL2(100 U/ml), IL4 (100 U/ml) or TUC2.15 SN (1% v/v) in the presence ofanti-IL2 receptor antibody 5A2 (30 ug/ml) or anti-IL4 antibody 11B11 (10ug/ml). Thymidine incorporation is measured on day 3.

                  TABLE 2                                                         ______________________________________                                        Growth of TS1 in Response to Various Cytokines                                Factors    Dose/Dilution                                                                              Cell Growth (A.sup.405)                               ______________________________________                                        TUC2.15 SN 1/12.500     1.96                                                  IL1        100 U/ml     0                                                     IL2        100 U/ml     0                                                     IL3        100 U/ml     0.01                                                  IL4        100 U/ml     1.36                                                  IL5        100 U/ml     0                                                     IL6        20 ng/ml     0                                                     GM-CSF     10 ng/ml     0                                                     G-CSF      4 ng/ml      0                                                     M-CSF (crude)                                                                            1/4          0.02                                                  EGF        50 ng/ml     0                                                     PDGF       4 ug/ml      0.02                                                  ______________________________________                                    

TS1 cells are incubated for 3 days in the presence in various factors orSN. All reagents are tested over a 100-fold range but results are givenfor the highest dose only. None of the factors that score negatively atthe highest dose have any effect at lower dose. Cell growth is measuredby calorimetric determination of hexosaminidase levels. Absorbance (A)of cultures at 405 nm incubated without growth factors ranges from about0.10 to about 0.15 and is subtracted.

EXAMPLE 3 Purification of the T Cell Growth Factor

Large batches of T cell SN are produced by stimulating TUC2.15 andTUC7.51 cells with ConA as described in Example 1. The active materialis concentrated by adsorption to silicic acid and applied to an UltrogelAcA54 gel filtration column. The major growth promoting activity, whichis destroyed by trypsin, elutes as a symmetrical peak in the 30-40 kDaregion (FIG. 2A), and is therefore designated P40. Subsequentexperiments are carried out with TUC7.51 SN because the concentrationsof P40 are higher in this material.

Preliminary characterization of the growth factor indicates that it hasa pI of ≈10 and is glycosylated, 60% of the activity being retained on alentil lectin column. Based in part on this information, the followingpurification protocol is adopted. Active fractions from the gelfiltration step are further separated by hydrophobic interactionchromatography on a TSK-phenyl column (FIG. 2B) followed by passagethrough a MonoQ anion exchange column equilibrated at pH 9.5. At thiselevated pH, most contaminants are retained on the column, whereasP40elutes mainly in the flow-through fractions, as expected from itshigh pI (FIG. 2C). Final purification is achieved by reversed phasechromatography on a C1-column equilihbrated with 0.05% (w/v) TFA. P40 isrecovered in a single peak eluting at an acetonitrile concentration of35% (v/v) (FIG. 2D). At the end of this purification, P40 stimulateshalf-maximal growth of TS1 at a concentration of ≈5 pg/ml (FIG. 3),which corresponds to a 2000-fold purification. On average, the overallyield ranges from about 5 to about 10%.

The purified protein is very heterogeneous with a Mr of about 32 toabout 39 kDa in NaDodSO₄ /PAGE both under reducing (FIGS. 4A-4B) andnon-reducing conditions. Biological activity is recovered from thecorresponding fractions of a non-reduced gel, but exposure to NaDodSO₄and 2-mercaptoethanol destroys most of the activity.

EXAMPLE 4 Amino Acid Sequence Analysis of Murine P40

Edman degradation of P40 (≈250 pmol) did not yield N-terminal sequence.For sequence analysis, P40 (immobilized on the polybrene-treated sampledisk of the sequencer) is acylated (Tarr, Methods of ProteinMicrocharacterization ed. J. E. Shively! Human Press, pp. 155-194, 1986)and then subjected to in situ cyanogen bromide treatment as described bySimpson I. Sequence analysis is then continued and yields the followingmajor amino acid sequence (110 pmol): NH₂ -Ala Gly Asn Thr Leu Ser PheLeu Lys Ser Leu Leu Gly Thr Phe Gln Lys Thr Glu.

This internal sequence show no significant similarity with that of otherproteins stored in the data bases listed in Example 1.

The determination of the complete amino acid sequence was achieved bychemical methods.

Briefly, before proteolytic digestion native P40 was reduced withdithiothreitol and carboxymethylated with iodoacetic acid to yieldCm-P40. Peptides (indicated in FIG. 7) were prepared for sequenceanalysis by cleavage of Cm-P40 with endoproteinase Asp-N, trypsin,chymotrypsin, and cyanogen bromide (denoted by D, T, C, and N,respectively in FIG. 7). Subpeptides of the endoproteinase Asp-N werederived by cleavage with Staphylococcus aureus V8 protease (denoted withhyphenated S suffixes). In FIG. 7, amino acid residues not identifiedare indicated by X.

The exact methodology of amino acid sequence determination is describedbelow:

A. Materials: Tween 20, guanidine hydrochloride (Sequenal grade) andtrifluoroacetic acid (F₃ AcOH; 99+% pure grade) were purchased fromPierce Chemical Co. (Rockford, Ill., USA). Iodoacetic acid (purissgrade) was obtained from Fluka (Buchs, Switzerland) and wasrecrystallized prior to use.

Dithiothreitol was from Calbiochem (La Jolla, Calif., USA). Sodiumchloride (Aristar grade) and acetic anhydride were purchased from BDH(Poole, UK). Cyanogen bromide (Univar grade) was from Ajax Chemical Co.(Sydney, Australia). All other chemicals were of the highest gradecommercially available.

Trypsin (treated with tosylphenylethylchloromethane) and chymotrypsinwere purchased from Worthington Biochemical Co. (New Jersey, USA).Staphylococcus aureus strain V8 protease was obtained from Miles Co.(Napperville, Ill., USA). Endoproteinase AspN from a Pseudomonas fragimutant and N-glycanase F were obtained from Boehringer Mannheim GmbH(West Germany). All organic solvents were HPLC grade (Chromar grade,Mallinckrodt, Ky., USA). Deionized water, obtained from a tandemMilli-RO and Milli-Q system (Millipore, Inc., Massachusetts, USA) wasused for all buffers.

B. Preparation of murine S-carboxymethyl-P40 (Cm-P40): P40 (15 ug in 120ul 35% aqueous acetonitrile containing 0.1% (v/v) F₃ AcOH and 0.02%Tween 20 was concentrated to approximately 10 ul by centrifugalcentrifugation (Savant, Ind. Hicksville, N.Y.), diluted to 160 ul with7.5M guanidine.HCL containing 0.2M Tris.HCl buffer, pH 8.5, 0.002M EDTAand 0.02% (v/v) Tween 20 and then reduced with dithiothreitol (0.015M)at 40° C. for 4.5 h. Alkylation was achieved by the addition ofiodacetic acid (final concentration, 0.05M) to the mixture andincubation continued for 30 min at 25° C. in the dark. The reaction washalted by addition of 25 ul of 2-mercaptoethanol. Cm-P40 was recoveredfrom the mixture using a reversed-phase high-performance liquidchromatography (RP-HPLC) procedure previously described (Simpson et al.,Eur J. Biochem. 1176:187-107, 1988, hereinafter Simpson II). TheCm-P40-containing fraction (60 ul) was adjusted to 0.02% (v/v) withrespect to Tween 20 and then diluted to 1 ml with an appropriate buffer(containing 0.02% (v/v) Tween 20) prior to enzymatic digestion.

C. Trypsin digestion: Cm-P40 (7 ug) in 1 ml of 1% (w/v) NH₄ HCO₃, pH 7.8containing 0.001M CaCl₂ and 0.02% (v/v) Tween 20 was digested with 0.5ug trypsin for 16 h at 37° C.

D. Chymotrypsin digestion: Cm-P40 (6 ug) in 1 ml of 1% (w/v) NH₄ HCO₃,pH 7.8 and 0.02% (v/v) Tween 20 was digested with 0.6 ug chymotrypsinfor 16 h at 37° C.

E. Endoproteinase Asp-N digestion: Cm-P40 (15 ug) in 1020 ul of 0.05Msodium phosphate buffer, pH 8.1, 0.02% (v/v) Tween 20 was digested with0.7 ug of freshly prepared endoproteinase Asp-N for 16 h at 34° C.

F. Staphylococcus aureus strain V8 protease digestion: EndoproteinaseAsp-N peptide D3 in 1 ml of 1% (w/v) NH₄ HCO₃, 0.02% (v/v) Tween 20 wasdigested with 0.5 ug S. aureus V8 protease at an enzyme/substrate ratioof 1:10 for 16 h at 30° C.

G. Purification of polypeptides by high-performance liquidchromatography instrumentation: Peptide mixtures resulting fromenzymatic cleavages were fractionated by reversed-phase HPLC on aHewlett-Packard liquid chromatograph (model 1090A) fitted with adiode-array detector (model 1040A) as described (Simpson II).

H. Column Supports: The following columns were used for the purificationof Cm-P40 and derived peptides: (a) Brownlee RP-300 (300 nm pore size,7-um particle diameter, octylsilica packed into a stainless steel column30×2.1 mm i.d. or 50×1 mm i.d. (Brownlee Laboratories, Santa Clara,Calif. USA). (b) Dimethylaminoazobenzene sulfonyl chloride (DABS-C1)amino acids were separated and quantitated on a Brownlee PTC amino acidanalysis column (220×2.0 mm i.d.) (Applied Biosystems, Foster City,Calif., USA).

I. Peptide nomenclature: The following prefixes are used to denote theorigin of various peptides: T, Trypsin; CN cyanogen bromide; C,chymotrypsin; D, endoproteinase Asp-N. Peptides resulting fromsub-digestion of endoproteinase Asp-N peptides with S. aureus V8protease are denoted by hyphenated S suffixes. Peptides are numbered inthe order of their positions in the final sequence.

J. Cyanogen bromide cleavage: After P40 (10 ug) was subjected to severalcycles of Edman degradation in the protein sequencer without anydetectable PTH-amino acids, the sequence analysis was stopped. In situcyanogen bromide cleavage of native P40 was performed on the glass fibersample disk of the protein sequencer according to a procedure previouslydescribed (Simpson, I). Sequencer background levels which had arisenduring the sequence analysis were reduced by treating the sample diskwith 30 ul aqueous 50% (v/v) N-ethylmorpholine followed by 10 ul aceticanhydride (60 min at 25° C.). The filter was vacuum dried and thentreated with at 20-fold excess of cyanogen bromide in 70% (v/v) formicacid for 15 h at 25° C. At the end of this time the sample filter wasvacuum dried for 30 min and the sequence analysis continued.

K. Amino acid sequence analysis: Automated Edman degradation of proteinand peptide was performed using Applied Biosystems sequencers (models470A and 477A) equipped with on-line phenylthiohydantoin (Pth) aminoacid analyzer (model 120A). Total Pth-amino acid derivatives from thesequencer were injected onto the liquid chromatograph using a modifiedsample transfer device as described Begg, et al., in "Techniques inProtein Chemistry", (Hugli, T. E., Ed.) Academic Press, Orlando Fla.,USA, in press!. Polybrene was used as a carrier.

EXAMPLE 5 Peptide purification by Microbore RP-HPLC

Cm-P40 (15 ug) was digested with endoproteinase Asp-N and the digestfractionated by RP-HPLC on a short microbore column (30×2.1 mm i.d.),employing a low-pH (F₃ AcOH, pH 2.1) mobile phase and a gradient ofacetonitrile. Three major peptide-containing peaks were detected: D1, D2and D3 (FIG. 8). Spectral analyses of these peptides were performedusing real-time photodiode-array spectroscopy and the absorption spectraof peptides D1, D2 and D3 are shown in FIG. 9. The high absorbance at290 nm of peptide D1 is indicative of the presence of a tryptophanresidue. The D2 and D3 peptides have high absorbance at 280 nm and lowabsorbance at 290 nm which is characteristic of tyrosine-containingpeptides. The presence of tryptophan residue in peptide D1 is supportedby the derivative absorbance spectrum shown in FIG. 10. Enhancement ofresolution by second-order-derivative spectroscopy reveals extrema at290; 2 nm and 280±2 nm which are characteristic of tryptophan residues.Peptide D3 was subdigested with S. aureus V8 protease and the resultantdigest fractionated by RP-HPLC at low pH (F₃ ACOH) (FIG. 11A).

Reversed-phase HPLC purification of peptides resulting from treatment ofCm-P40 with chymotrypsin and trypsin were performed and analyzed in asimilar manner. Reversed-phase fractionation of these digests, however,resulted in a complex pattern of peptide-containing peaks (FIGS. 11B and11C). All of the peptide fractions from the first dimension RP-HPLC weresubjected to a second chromatographic step using the samechromatographic support and acetonitrile gradient but a different mobilephase (e.g., unbuffered sodium chloride or 20 mM sodium phosphate, pH7.0). For some peptides a third chromatographic step was necessarybefore a homogeneous peptide could be isolated. In the latter situation,an ODS-hypersil column and a different organic solvent (methanol) wereused for the chromatography (Simpson II).

EXAMPLE 6 Characterization of Glycosylation State of P40

Cm-P40 (0.5 ug) was iodinated using the iodine monochloride procedure.¹²⁵ I-Cm-P40 was separated from free ¹²⁵ I by sequential gel filtrationand cation-exchange chromatography. ¹²⁵ I-Cm-P40, untreated, reducedwith 2-mercaptoethanol for 5 min at 95° C., or digested with N-glycanaseF (Genzyme, Boston, Mass., USA) or endo-α-N-acetylgalactosaminidase(O-glycan-peptide hydrolase, Boehringer Mannheim) for 16 h at 37° C.according to the manufacturer's instructions was electrophoresed on a10-15% gradient polyacrylamide gel in the presence of SDS. The gel wasstained with Coomassie Blue R250 using the Phast electrophoresis system(Pharmacia, Uppsala, Sweden) according to the manufacturer'sinstructions. ¹²⁵ I-Cm-P40 was detected by autoradiography usingHyperfilm, MP (Amersham, Buckinghamshire, UK).

Purified iodinated P40 electrophoresed as a single broad band ofapparent M_(r) 32,000-39,000 daltons on 12% SDS-PAGE in both theunreduced and reduced (2-mercaptoethanol) state (see FIG. 4B Lane 1 and4) indicating that it is a monomeric protein.Endo-α-N-acetylgalactosaminidase (O-glycanase) treatment of P40 had noapparent effect on the molecular mass (FIG. 4B, Lane 3) but treatmentwith N-glycanase F caused a reduction in apparent M_(r) to 15,000-16,000Da (FIG. 4B, Lane 2). The lack of effect of O-glycanase indicates thatP40 does not contain O-linked carbohydrate chains or that these sitesare not accessible in the intact molecule. Since N-glycanase F releasescarbohydrate moieties attached to asparagine residues (N-linked) thisindicates that P40 consists of a protein core (M_(r) 15,000-16,000) withconsiderable amounts of N-linked sugars.

Murine P40 has 126 amino acids. The calculated M_(r) from the sequenceanalysis is 14,150. The difference in the calculated M_(r) and themeasured M_(r) for native P40 (32-39 kDa) can be attributed toN-glycosylation since upon treatment with N-glycanase F the M_(r) isreduced to 15,000-16,000 (FIG. 4). The protein sequence data providesinformation on the post-translational processing of mature P40. Forinstance, since no amino acid was identified at positions 32, 60, 83 and96 (FIG. 10) and since these positions meet the criteria for N-linkedglycosylation sites (i.e. Asn-Xaa-Thr/Ser), these data are consistentwith asparagines being glycosylated at these four positions.Confirmation of asparagine residues at positions 32, 60, 83 and 96 andthe COOH--terminal residue (P40-126) was provided by sequence analysisof a P40 cDNA clone.

EXAMPLE 7 Analysis of N-terminal Block in Murine P40

The exact nature of the blocked N-terminus was determined by acombination of amino acid analysis, fast-atom-bombardment massspectrometry, and peptide synthesis. These analyses indicated theN-terminus of murine P40 is likely to be pyroglutamic acid.

The methods involved in determining the amino terminal residue of P40and a discussion thereof follow:

Method

A. Amino acid analysis: Amino acid analysis was performed on a Beckmanamino acid analyzer (model 6300) equipped with a model 7000 data systemor by using the dimethylaminoazobenzene sulfonyl chloride (DABS-C1)precolumn derivatization procedure using microbore column RP-HPLC(Simpson, et al., Euro. J. Biochem. 153: 629-637, 1985). Samples werehydrolyzed in vacuo at 110° C. for 24 h with gaseous HCl generated from6M HCl containing 0.1% (w/v) phenol.

B. Fast-atom-bombardment mass spectrometry: Fast-atom-bombardment massspectrometry (Barber et al., Anal. Chem., 54: 645A-657A, 1982) wasperformed using a VG 70/70E-HF forward-geometry double-focussing massspectrometer (VG Analytical, Manchester, UK) equipped with an Ion Techsaddle-field fast-atom gun. Sample was applied in 2 ul of 0.1% (v/v)aqueous F₃ AcOH to the sample stage containing 1 ul of pre-appliedmixture of dithiothreitol/dithioerythritol (5:1). Xenon atoms at apotential of 8 keV and a discharge potential of 1 mA were used forsample bombardment. Scans were performed at 40s/decade at a resolutionof 1500. Positive-ion spectra were acquired by multi-channel analysismode using a VG 11/250J data system.

C. Peptide synthesis: Fluorenylmethoxycarbonyl (Fmoc)polyamide solidphase peptide chemistry was employed to synthesize two peptidescorresponding to the N-terminal decapeptide of P40 (endoproteinase Asp-Npeptide D1) with either glutamic acid or glutamine as the amino terminalresidue. The conventional Fmoc polyamide side chain protecting groupswere employed: Trp (CHO); Arg (Mtr),Mtr=4-methoxy-2,3,6,-trimethylbenzenesulfonyl); Glu (OtBu); Cys (tBu).Pentafluorophenyl (OPfp) esters of all Fmoc-amino acids indimethylformamide with the exception of Fmoc-Arg (MtC) which wasactivated as an 1-hydroxybenzotriazole (HOBt) ester, were employed forsequential coupling of activated amino acids on RaMPS (DuPont, N.J.)Wang resin. Peptide bond formation was generally complete within 120 minprovided that the concentration of the OPfp ester was 2.5×times greaterthan the concentration of derivatized resin and provided that oneequivalent of 1-hydroxybenzotriazole was added to the coupling mixturein order to catalyze the reaction. Upon completion of the synthesis, thepeptide was deprotected and cleaved from the resin by extended treatmentwith F₃ ACOH (containing 5.4% thioanisol, 0.6% 1,2-ethanedithiol). Crudesynthetic peptides and their derivatives (e.g. S-carboxymethyl-peptides)were purified by reversed-phase HPLC. The pure synthetic peptideschromatographed as single peaks on reversed-phase HPLC (Brownlee RP-300column 30×2.1 mm, i.d.) and gave the expected amino acid ratios.

Peptides (30 ug in 100 ul 0.1% F₃ ACOH) were acetylated with aceticanhydride by treatment with 6 ul N-ethylmorpholine (Pierce, sequenalgrade) followed by 2 ul acetic anhydride (Fluka, puriss grade) for 10min at 25° C. Formation of pyroglutamyl peptides was accomplished bytreating the glutamine peptide at 110° C. for 16 h at pH 7.8 undernitrogen.

Discussion

Analysis of three major Asp-N peptides (D1-D3) and the S. aureus V8protease subpeptides of D3 provided 65% of the P40 amino acid sequence.Of the three peptides, D1, the single tryptophan-containing peptide wasN blocked, indicating that this peptide was derived from the N-terminalportion of the polypeptide chain. The amino acid composition of theN-blocked Asp-N peptide D1 was consistent with the tryptophan-containingtryptic peptide T1 with the N-terminal addition of two extra residues(Glx and Arg).

Fast-atom-bombardment mass spectrometry (FAB-MS) of Asp-N peptide D1revealed a protonated molecular ion (MH⁺) of mass 1248 which onlycorresponds to the amino acid composition of this peptide if theblocking group was assumed to be pyroglutamic acid. The nature of theN-blocking group was examined using two synthetic decapeptides D1 witheither glutamic acid or glutamine at the amino terminus (see FIGS. 7 and12). As shown in FIG. 12, the endoproteinase Asp-N peptide D1 and thepyroglutamyl synthetic peptide co-chromatographed on reversed-phase HPLC(retention time, 20.50±-0.2 min) and were well resolved from theglutamyl synthetic peptide (retention time, 22.70±0.2 min). Afteracetylation, the chromatographic behavior of D1 and the pyroglutamylsynthetic peptide was identical (no increase in retention time) whilethe acetylated glutamyl synthetic peptide exhibited a marked increase inretention behavior (retention time was 28.97±0.2 min compared to thenon-acetylated form, 22.70±0.2 min). The amino terminus of P40 is likelyto be pyroglutamic acid since the amino-terminal endoproteinase Asp-Npeptide D1behaves in exactly the same manner as the syntheticpyroglutamyl peptide before and after acetylation on reversed-phaseHPLC. FAB-MS of the glutamyl synthetic peptide yielded a molecular massof 1248 which was in perfect agreement with that obtained for Asp-Npeptide D1.

EXAMPLE 8 Biological Activity of Purified P40

Purified P40, at concentrations up to 20 ng/ml, did not support theproliferation of either IL3-dependent myeloid cell lines (FDCP-1, Ea3.15and DA-1), IL5-dependent B cell lymphoma BCL1, or IL6-dependent B cellhybridoma 7TD1. Unlike IL2, and to some extent IL4, it also fails tostimulate any of six cytolytic T cell clones tested (Table 3) Bycontrast, strong proliferations are observed with some but not allhelper T cell lines. Both IL2-producing (TH₁ type, TUC7.33) andIL4-producing (TH₂ type, e.g., TUC2.15) clones are found among theresponders. A significant correlation, illustrated in Table 3 for cloneTUC7.51, is observed between the time spent in culture and the responsesto P40 and IL4.

                  TABLE 3                                                         ______________________________________                                        Comparison of the T Cell Growth Factor Activities of                          IL2, IL4, and Purified P40                                                                     Proliferation in response to.sup.a                           T Cell Lines and Clones                                                                         IL2       IL4     P40                                       ______________________________________                                        Cytolytic T Cells                                                                          CTLL-2   445       0.6   0.8                                                  P35:10   683       24    1.2                                                  P35:48   303       4     1.8                                                  P91:6    195       2     1.6                                                  P1:5     993       12    1.2                                                  P1:204-8 630       9     1.2                                     Helper T Cells                                                                             TUC2     311       2     14                                                   TUC2.15  1,806     813   240                                                  TUC5     263       20    13                                                   TUC5.37  253       4     0.8                                                  TUC7.33  115       276   235                                                  TUC7.51.sup.b                                                                          1,179     27    17                                                   TUC7.51.sup.c                                                                          1,345     634   199                                                  TUC13.1  116       51    1.7                                     ______________________________________                                         A)Cells are incubated with or without the indicated growth factors and        thymidien incorporation is measured on day 3. Factor dosage is as follows     100 U/ml for IL2 and IL4, and 10.sup.3 -10.sup.4 U/ml for P40. The result     are shown as ratios of radioactivity incorporated with and without            factors.                                                                      B)1 monthold culture                                                          C)1 yearold culture                                                      

EXAMPLE 9 Cloning and Characterization of the Murine P40 Gene

Screening of CDNA library. Double-stranded CDNA was prepared accordingto Gubler, et al., Gene 25:263, 1983, using polyadenylated RNA isolatedfrom P40-producing helper T cells TUC7.51 after a 24 h stimulation withConA (2.5 ug/ml). The CDNA was cloned into the BamHI site of a pUC8vector and transformed into E. Coli strain DH5. Transformants werescreened by in situ hybridization with two end-labeled 20-meroligonucleotide probes. For the initial screening, a 64-fold degenerateprobe

5'-TGCAT(C+T)TC(X)GT(C+T)TT(C+T)TG(G+A)AA-3'! corresponding to aminoacid sequence FQKTEMQ (positions 114-120, see text) was used. Positiveclones were subsequently tested with a 129-fold degenerate probe

5'-GG(A+G)TC(A+G)TC(T+C)TT(X)AG(A+G)TT(C+T)TC-3'! corresponding tosequence ENLKDDP (positions 17-23, see text).

DNA sequencing. DNA was sequenced by the dideoxynucleotide procedureafter subcloning into a M13 vector. Appropriate fragments were generatedby digestion with Pst1 and Nco1 restriction endonucleases using thesequencing strategy shown in FIG. 5.

Characterization of cDNA and Construction of Expression Vectors. A cDNAlibrary was prepared, in a pUC8 vector, from a helper T cell clone thatproduces large amounts of P40 after stimulation with ConA. This librarywas screened with two oligonucleotide probes synthesized on the basis ofselected amino acid sequence data obtained by analysis of P40 peptides.Of 20,000 independent transformants, 112 hybridized with the two probes.Most of these clones contained cDNa inserts of about 500 bp. Using oneof these cDNA's as a probe, a strong signal was obtained with atranscript of about 700 nucleotides in Northern blots of poly(A)⁺ RNAisolated from P40-producing helper T cell clone TUC7.51. Poly(A)⁺ RNAfrom P815 mastocytoma, which does not produce detectable P40 activity,gave no signal at all.

To establish that the selected clones contained authentic P40 cDNA, anexpression vector was constructed. Insert P40.2B4 was cloned into BamHIsite of plasmid pZIPneoSV(X)1 (Cepko, et al., Cell 37: 1053, 1984) andtransfected into Clone-1d fibroblasts (Kit, et al., Exp. Cell. Res. 31:297, 1963). Cell supernatants collected 48 h after transfection weretested for their growth factor activity on P40-dependent TS1 cells. Asshown in FIG. 6, supernatants from cells transfected with P40.2B4 cDNA(closed symbols), but not from mock-transfected cells (open symbols),supported the growth of TS1. This result indicates that P40.2B4 cDNApresumably contains the entire coding region of P40.

The complete nucleotide sequence of the cDNA insert of clone P40.2B4 wasdetermined, and is shown under "Detailed Description of the Invention".It consists of 554 nucleotides with a 5' untranslated sequence of 15nucleotides, an open reading frame of 432 nucleotides and a 3'untranslated region of 107 nucleotides. The 3'-end terminates with astring of 18 adenine residues located 12 nucleotides downstream from anAATAAA polyadenylation signal consensus sequence. The 3'untranslatedregion contains 3 copies of the sequence ATTTA which is characteristicof transiently expressed genes such as GM-CSF, G-CSF, interferons,several interleukins, tumor necrosis factor, and oncogenes c-fos andc-myc (Shaw et al., Cell 46: 659, 1986); two of these repeats atnucleotide positions 461-468 and 470-477 are part of an 8 nucleotidemotif TATTTATT, which is also present in many of these molecules (Caputet al., Proc. Natl. Acad. Sci. USA 83: 1670, 1986).

The predicted polypeptide encoded by the CDNA insert of clone P40.2B4consists of 144 residues. This size estimation is based on thepresumption that the first ATG in the sequence (nucleotide position16-18) is the initiator codon, a view supported by the efficientexpression of the cDNA in fibroblasts and by the presence of an adenineat nucleotide position 13, in concordance with the consensus sequencefor an initiator ATG codon; an in-frame TGA translation terminationcodon occurs as nucleotides 448-450. The deducted P40 sequence ischaracterized by a hydrophobic N-terminal seugence typical of a signalpeptide. Because of the presence of a blocked N-terminus in the nativeprotein, there is some uncertainty concerning the N-terminal residue.Based on the probability weight-matrix described, (Von Heijne, NucleicAcid Res. 14:4683, 1986), the most likely N-terminal sequence of themature protein would be Gln-Arg-Cys. This is consistent with evidenceobtained by biochemical analysis of P40 peptides. Mature P40 would thenconsist of 126 amino acids with a predicted relative molecular mass of14,150. The difference with the Mr measured for native P40 appears to bedue to glycosylation as suggested by the presence of 4 potentialN-linked glycosylation sites and confirmed by the about 15 kDa Mr of thenative protein after N-glycanase-treatment. The sequence of P40 isfurther characterized by the presence of 40 cysteines and a strongpredominance by cationic residues, which explains the elevated pI(10) ofthe native protein.

EXAMPLE 10 Isolation of the Human Genomic P40 Gene

The human genomic P40 gene was cloned using the murine P40 CDNA clone asa probe. Briefly, a human genomic library was constructed in phageλGEM11 (Promega, Madison, Wis.) with Sau3A-cut DNA isolated from anEBV-immortalized human lymphoblastoid cell line (CESS). The library wasscreened with a ³² P-labeled mouse P40 cDNA under low stringencyhybridization conditions (two washes at 55° C. in 2x SSC and 0.1% SDS)and yielded a positive clone (λH40.3a1). Analysis of this cloneidentified a 900 bp HindIII fragment which encoded a region of highhomology with the 3' region of the murine cDNA.

Overlapping restriction fragments from λH40.3a1 were subcloned into M13vectors and sequenced by the dideoxynucleotide method. A 2kb SmaI/BamHIfragment was sequenced using deletion mutants generated by digestionwith exonuclease III as described by Henikoff, S. (1984) Gene 28:351.FIG. 13 depicts a restriction map of the human gene; its nucleotide andamino acid sequence are shown in FIG. 14.

EXAMPLE 11 Isolation of a Human P40 CDNA

A human CDNA library constructed in λgt10 using poly(A)⁺ RNA preparedfrom peripheral blood mononuclear cells (PBMC) stimulated for 24 h withphytohemagglutinin (PHA), 30 ug/ml, and phorbol myristate acetate (PMA),100 ng/ml. The library was screened with a 0.9-kb HindIII restrictionfragment of the human genomic clone described in Example 10. Filterswere washed at 65° C. with 30 mM NaCl, 3 mM sodium citrate and 0.1% SDS.Screening yielded two positive clones, λcH40.2 and λcH40.4. The DNAsequence of the human P40 CDNA was deduced by dideoxynucleotidesequencing of these two clones. The nucleotide and amino acid sequencesare shown under "Detailed Description of the Invention".

The human P40 cDNA has an open reading frame of 432 nucleotidesspecifying a polypeptide of 144 amino acids, which includes, by homologywith the mouse protein, a presumptive signal peptide of 18 residues. Thepredicted molecular mass of the unglycosylated mature protein is 14,110Daltons. As for mouse P40, the presence of four potential N-linkedglycosylation sites makes it likely that the molecular mass of thenative protein is much more elevated. The homology with mouse P40 is 69%at the nucleotide level and 55% at the protein level. Noteworthy, the 10cysteines in the mature protein are perfectly conserved but human P40has an additional cysteine in the presumptive signal peptide. No otherhomologies were detected between human P40 and previously sequencedproteins.

The size of the RNA for human P40 was determined in Northern blots ofpoly(A)⁺ RNA isolated from PBMC stimulated with PHA (0.5%) and PMA (100ng/ml) for 24 hours. P40 CDNA hybridized with a band of ≈700 bp, a sizecomparable to that of the mouse P40 message. No message was observed infreshly isolated unstimulated PBMC.

EXAMPLE 12 Isolation of the Murine Genomic P40 Gene

The murine genomic P40 gene was cloned using the murine P40 cDNA cloneas a probe. A subgenomic library was constructed in phage λGEM11 withsized fragments isolated by agarose gel electrophoresis fromBamHI-digested high molecular weight DNA of murine L1210 leukemic cells.Screening with a ³² P-labeled mouse P40 cDNA yielded a positive clone(λS40.1a) which was sequenced by the dideoxynucleotide method usingoverlapping restriction fragments cloned in M13 vectors. FIG. 13 depictsa restriction map of the murine P40 genomic gene; its nucleotide andamino acid sequence are shown in FIG. 15.

EXAMPLE 13 Comparison of Human and Murine Genomic Genes

As indicated in FIG. 13 the murine and human genomic genes share anidentical organization: they both are approximately 4 kb in length andconsist of 4 introns and 5 exons flanked by consensus donor and acceptorsplice sites. The complete sequence of the two genes is shown in FIGS.14 and 15.

Although the size of each exon is identical in the two genes, theirhomology is somewhat variable with a minimum of 60% in the first and amaximum of 76% in the last exon. This results in a 55% homology at theamino acid level and in a perfect conservation of the 10 cysteineresidues present in the two mature proteins. The introns are also verysimilar in size, except for intron 4, which is about 0.5 kb longer inthe human gene. However, the introns show no significant homology exceptfor intron 2, which is 69% identical in the two species. A stretch ofalternating purines and pyrimidines is found in the third mouse intron(poly dA-dC) and in the fourth human intron (poly dG-dT). Thesesequences have a strong potential to adopt a left-handed conformation(Z-DNA), and reportedly display an enhancer-like activity Hamada, H.(1984) Mol. Cell. Biol. 4:2622!. In the fourth intron of the human gene(nucleotides 2200-2500), an Alu sequence was identified.

The 5' flanking regions of mouse and human P40 genes contain longstretches of highly conserved sequences, indicating that the homologybetween the two genes is not restricted to the coding regions (FIG. 16).Both genes have a TATA-like sequence, located 57 and 56 bp upstream theATG start codon, respectively. Several consensus motifs conceivablyinvolved in the transcriptional control are well conserved in the twogenes. Upstream of the TATA box, is one copy of the consensus bindingsite for AP-1, a factor involved in the regulation of genes by phorbolesters Lee, W. et al. (1987) Cell 49:741!. This sequence (5'-TGACTCA-3')is likely to be functional in the P40 gene inasmuch as phorbol estersstrongly enhance P40 expression. In both the human and the mouse genes,the AP-1 binding consensus sequence partly overlaps with a copy of theinterferon regulatory factor-1 binding element (5'-AAGTGA-3') Miyamoto,M. et al. (1988) Cell 54:903!. Farther upstream, is noted a C-richmotif, that is present in the AP-2 binding site of the SV40-enhancerMitchell, P. J. (1987) Cell 50:847!. Functional studies of the promotercan determine what role the latter sequences play in the regulation ofP40 expression.

The 3' untranslated regions of human and mouse P40 also show significant(63%) homology (FIG. 16). The human gene contains four copies of theATTTA sequence, which is frequently found in other transiently expressedcytokines and apparently reduces RNA stability Shaw, G. et al. (1986)Cell 46:659!. Three copies of this motif are conserved in the mousegene. By contrast, the polyadenylation signal consensus sequences arenot conserved.

S1 nuclease mapping of the mouse gene identified a major start oftranscription centered 33 bp upstream of the ATG start codon and 24 bpdownstream of the TATA-like sequence. In the human P40 gene, the mainstart of transcription was localized approximately 30 bp upstream of theATG start codon and 26 bp downstream of the TATA-like sequence.

Southern blots of mouse DNA isolated from two distinct cell lines andprobed with homologous P40 cDNA under high stringency showed thehybridization pattern expected from the restriction cleavage map of theP40 gene, indicating that P40 is a single copy gene in the haploidgenome. Similar results were obtained for the human gene. Moreover,extensive comparisons carried out for each P40 exon and intron failed toidentify significant homologies with recorded genes.

EXAMPLE 14 Expression of the P40 Gene in Human PBMC

No message for P40 could be detected in unstimulated PBMC butstimulation of the cells with PMA and a calcium ionophore induced strongexpression of a 0.7 kb P40 mRNA that became detectable after 8 hours andreached a maximum level after ≈24 hours (FIG. 17). P40 thus appears tobelong to the growing family of cytokines that are not constitutivelyexpressed in normal PBMC but are rapidly induced after activation of thecells.

In order to examine whether P40 expression could be induced with otherstimuli, we exposed PBMC to LPS or Staphylococcus aureus strain Cowan 1.No message was detected under these conditions (FIG. 18), suggestingthat the P40 gene is not readily induced in monocytes and in B cells. Bycontrast, exposure to T cell mitogens such as PHA or anti-CD3 antibodiesinduced a significant P40 response. Since anti-CD3 antibodies areconsidered to mimick antigenic stimulation, it is likely that P40 issecreted by normal T cells in the course of their physiological responseto antigen. Addition of PMA to these T cell mitogens further increasedthe levels of P40 mRNA although PMA by itself had no detectable effecton P40 expression. It is unlikely that this synergistic effect of PMA isdue to induction of P40 in non-T cells because of the absence of P40message in non-rosetting PBMC (FIG. 19). Therefore, these observationsseem to imply that optimal P40 expression by T cells requires bothprotein kinase C activation and increased intracellular calcium levels.

To investigate whether P40 is elaborated by a particular subset of Tlymphocytes, T cells were purified by nylon wool filtration and CD4⁺cells separated from CD8⁺ by fluorescence-activated cell sorting. Asshown in FIG. 19, P40 was preferentially expressed in the CD4⁺ subset.These results together with observations made in the mouse (Example 8)indicate that, in the immune system, P40 is essentially produced byhelper T cells.

EXAMPLE 15 Expression and Biological Activity of Recombinant P40

A. Baculovirus expression system and semi-purification of recombinantP40:

Recombinant human P40 was expressed in Spodoptera frugiperda S49 cellsusing a recombinant baculovirus. A similar expression and biologicalactivity system was used for recombinant murine P40,

In the case of human P40, a 489 bp EcoRI-HindIII fragment of λcH40.4 wassubcloned into the BamHI site located in the 5' end of the polyhedringene in plasmid pVL941 Luchow, V. A. et al. (1989) Virology 170:31!.This construct was transfected along with DNA of Autographa californicanuclear polyhedrosis virus into Spodoptera frugiperda S19 cells andrecombinant baculovirus clones were isolated by limiting dilution anddot hybridization as described Summers, M. D. (1988) A Manual of Methodsfor Baculovirus Vectors and Insect Cell Culture Procedure. TexasAgricultural Experiment Station Bulletin No. 1555!. Expression of therecombinant protein was verified in infected cells incubated with ³⁵S-labeled methionine. A P40-enriched fraction was obtained by adsorptionof cationic proteins on sulfopropyl-Sepharose after dilution with 5volumes of 33 mM sodium acetate, pH 5. Adsorbed proteins were elutedwith 0.9M NaCl, 0.1M Tris-HCl, pH 8 and Tween-20 (10⁻⁴ vol/vol),precipitated with 10% trichloroacetic acid, and analyzed by SDS-PAGE.The recombinant protein used in the T-cell survival assay wassemi-purified by adsorption to and elution from sulfopropyl-Sepharoseunder similar conditions and dialyzed against phosphate-buffered salinebefore use. The recombinant protein had an M_(r) of ≈23 kDa (FIG. 4)similar to that of mouse P40 and probably reflecting N-linkedglycosylation. In the case of murine P40, N-glycanase treatment reducesthis M to the predicted 14 kDa.

B: Biological Activity of Recombinant Human P40:

Mouse P40 was originally identified by its capacity to induce long-termproliferation of certain murine helper T cell lines. Subsequentexperiments have shown that a more frequent effect of P40 is to increasecell survival without inducing cell proliferation. Therefore thebiological activity of recombinant human P40 was examined for a capacityto enhance the survival of a number of human T cell lines that had beenmaintained in culture in the presence of PHA, irradiated PBMC asfeeders, and IL-4.

Stimulations were performed for 24 hours in RPMI 1640 mediumsupplemented with 10% fetal calf serum. Long-term T cell lines werederived from PBMC of hemochromatosic patients by repeated weeklystimulation with irradiated (3000 rads) allogeneic PBMC, PHA-P (30μg/ml) and recombinant human IL4 (25 U/ml). Prolonged survival wasobserved with two out of 10 lines tested, demonstrating the functionalintegrity of recombinant human P40 expressed in insect cells (FIG. 20).It is of interest that mouse P40 also enhanced the survival of the humanT cell lines, whereas human P40 was apparently not active on mouseP40-dependent cell lines.

EXAMPLE 16 Synergistic Effect of P40 and IL4 or IL3

The proliferative effects of co-culturing helper T cells in the presenceof P40 and IL4 or IL3 was investigated. TUC2.15N and TUC7.41 cells(5×10⁴ /well) were cultured with suboptimal amounts of P40 in thepresence or absence of a suboptimal dose of IL4 or close to optimal doseof IL3. After 3 days in culture, the cells were pulsed with tritiatedthymidine. The results in Table 4 indicate that the helper T cellstreated with P40 and IL4 or P40 and IL3 incorporated from about 4-40times more thymidine than those cells treated with any one of theseproteins. Thus, a strong synergy between P40 and IL4 or IL3 exists withrespect to stimulating proliferation of helper T cell lines that respondto these proteins.

                  TABLE 4                                                         ______________________________________                                        Synergism between P40 and IL4 or IL3                                                  P40     IL4      IL3   Thymidine Incorporation                        Cells   (U/ml)  (U/ml)   (ng/ml)                                                                             (cpm)                                          ______________________________________                                        TUC2.15N                                                                               0      0        0      122                                                   80      0        0     1296                                                    0      20       0     4446                                                   80      20       0     19248                                                   0      0        3     4781                                                   80      0        3     54366                                          TUC7.51N                                                                               0      0        0      120                                                   80      0        0     4958                                                    0      20       0     4459                                                   80      20       0     31050                                                   0      0        3     4354                                                   80      0        3     72157                                          ______________________________________                                         TUC2.15N and TUC7.51 helper T cells (5 × 10.sup.4 /well) were           cultured with suboptimal amounts of P40 in                                    the presence or absence of a suboptimal dose of IL4 or of an optimal dose     of IL3. The cultures were                                                     pulsed with tritiated thymidine after 3 days.                            

What is claimed is:
 1. A method of preparing mammalian T cell growthfactor P40, which supports interleukin 2- and interleukin 4-independentgrowth of T cells, comprising contacting a helper T cell or peripheralblood mononuclear cell culture with a T cell specific mitogen for aperiod of time and under conditions sufficient to induce production ofsaid P40, and recovering said P40.
 2. The method of claim 1 wherein saidrecovering step comprises contacting a supernatant of said culture witha hydrophobic chromatography resin, identifying and recovering a firstfraction therefrom having P40 activity, contacting said first fractionwith an ion exchange chromatography resin, identifying and recovering asecond fraction therefrom having said activity, contacting said secondfraction with a reverse-phase HPLC resin and identifying and recoveringa third fraction therefrom having said activity whereby said thirdfraction is homogeneous P40.
 3. The method of claim 2 wherein saidhydrophobic resin is TSK-phenyl chromatography resin, said ion exchangeresin is Mono-Q chromatography resin and said reverse-phase resin is C1reversed-phase HPLC resin.
 4. The method of claim 2 wherein saidactivity is stimulating interleukin 2- and interleukin 4-independentgrowth of helper T cells.
 5. The method of claim 1, wherein said P40 ismurine P40.
 6. The method of claim 1, wherein said P40 is human P40. 7.The method of claim 1, wherein said P40 comprises the amino acidsequence set forth in FIG.
 14. 8. The method of claim 1, wherein saidP40 comprises the amino acid sequence as set forth in FIG. 15.